9057024896

Vascular Endothelium in Human Physiology
and Pathophysiology
The Endothelial Cell Research Series
A series of significant reviews of basic and clinical research related to the endothelium.
Edited by Gabor M.Rubanyi, Berlex Biosciences, Richmond, California.
Volume One
Endothelium-Derived Hyperpolarizing Factor
edited by Paul M.Vanhoutte
Volume Two
Endothelial Modulation of Cardiac Function
edited by Malcolm J.Lewis and Ajay M.Shah
Volume Three
Estrogen and the Vessel Wall
edited by Gabor M.Rubanyi and Raymond Kauffman
Volume Four
Modern Visualisation of the Endothelium
edited by J.M.Polak
Volume Five
Pathophysiology and Clinical Applications of Nitric Oxide
edited by Gabor M.Rubanyi
Volume Six
Mechanical Forces and the Endothelium
edited by Peter I.Lelkes
Volume Seven
Vascular Endothelium in Human Physiology and Pathophysiology
edited by Patrick J.T.Vallance and David J.Webb
Volume in Preparation
Morphogenesis of Endothelium
W.Risau
This book is part of a series. The publisher will accept continuation orders which may be
cancelled at any time and which provide for automatic billing and shipping of each title in
the series upon publication. Please write for details.
Vascular Endothelium in Human Physiology
and Pathophysiology
Edited by
Patrick J.T.Vallance
Centre for Clinical Pharmacology

University College London Medical School
UK
and
David J.Webb
Clinical Pharmacology Unit and Research Centre

University of Edinburgh
UK
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ISBN: 90-5702-489-6 (Print Edition)
ISSN: 1384-1270
CONTENTS
Foreword ix
Sir John Vane
Contributors xi
Acknowledgements xv
1 Endothelial Nitric Oxide 3
Aroon D.Hingorani and Patrick J.Vallance
2 The Endothelin System: Physiology 31

Alan J.Bagnall and David J.Webb
3 Cyclooxygenase-dependent Endothelium-derived Contracting Factors 63

Stefano Taddei, Agostino Virdis, Lorenzo Ghiadoni and Antonio Salvetto
4 Endothelium-derived Hyperpolarizing Factor 75

Michel Félétou and Paul M.Vanhoutte
5 Regulation of Blood Cell Function by Endothelial Cells 95

Marek W.Radomski and Anna S.Radomski
6 Endothelial Dysfunction and Hypertension 109

Roberto Corti, Isabella Sudano, Christian Binggeli, Eduardo Nava,
Georg Noll and Thomas F.Lüscher
7 Endothelial Control of Vascular Tone in Chronic Heart Failure 131

Wolfgang Kiowski and Helmut Drexler
8 Hypercholesterolemia, Atherosclerosis, and the NO Synthase Pathway 147

John P.Cooke and Mark A.Creager
9 Endothelial Function in Diabetes Mellitus 173

Jim M.Ritter, Phil J.Chowienczyk and Giovanni E.Mann
vii
viii Contents
10 Flow-mediated Dilatation and Cardiovascular Risk 191

David S.Celermajer and John E.Deanfield
11 Endothelial Mediators and Renal Disease 205

Raymond J.MacAllister and William B.Haynes
12 Infection and Vascular Inflammation 227

Kiran Bhagat and Timothy W.Evans
13 The Endothelium in Human Pregnancy 247

Lucilla Poston and David J.Williams
Index 283
FOREWORD
Once thought to represent an inert lining to the blood vessels, an ever increasing number
of crucial functions of the vascular endothelium have emerged in recent years. This
monolayer of cells which envelops the circulating blood maintains patency of the vessel
through the production of anti-platelet and anti-thrombotic agents. It also regulates vascular
tone through the generation of the vasodilator agent, nitric oxide, and the potent
vasoconstrictors, endothelin-I and angiotensin II. The endothelium is a signal transducer
for vascular effects of circulating hormones as well as a site for metabolism of hormones
and blood lipids.
Arising from the discovery of the central role of normal endothelial function in
maintaining cardiovascular homeostasis, has emerged the concept that endothelial
dysfunction may contribute importantly to cardiovascular disease. Reduced nitric oxide
function has been recognised in a wide range of conditions associated with a predisposition
to cardiovascular events, including hypertension, hypercholesterolaemia, diabetes, the
menopause and heart failure, and in a wide range of vessel types including peripheral
resistance vessels, large conduit arteries and the coronary circulation. In some instances
the ‘fault’ seems to be in the pathway generating nitric oxide. However, in others, increased
degradation of nitric oxide, perhaps as a result of oxidative stress, may play an important
role. In either case the end result is a tendency to increased vascular tone, increased risk
of vascular thrombosis, and initiation or potentiation of the atherogenic process. More
recently it has become clear that there is a close relationship between the endothelin and
nitric oxide systems, and in many situations where the nitric oxide system is underactive,
the activity of the endothelin system is enhanced, compounding the adverse effects on
vascular structure and function. Indeed, a more ‘global’ dysfunction of the endothelium,
affecting a number of endothelial mediators, may be a feature of many forms of
cardiovascular disease.
In recent years, vascular biology, and in particular the biology of the endothelium, has
been an exciting and fast moving area of research. The present understanding of the role of
the endothelium in cardiovascular health and disease resulted from the rapid application of
the results of animal experiments to studies in patients. Endothelial dysfunction is now
seen as perhaps one of the earliest markers of vascular disease and an important target for
therapeutic intervention. Novel drug approaches have been made possible by the
development of new agents targeting the nitric oxide system, including L-arginine and the
nitrosothiol nitric oxide donors, the widespread potential for use of anti-oxidant vitamins,
and the very rapid clinical development of endothelin receptor antagonists.
ix
x Foreword
Now that the concept of endothelial dysfunction is widely accepted, and the key issues
can be addressed by mechanistic studies and clinical trials in patients, it is timely to draw
together our understanding of the role of endothelial function in health and cardiovascular
disease. This book, involving state-of-the-art reviews by leaders in their respective fields,
provides an up-to-date review of the vascular functions of the endothelium and its role in
key areas of cardiovascular disease in humans. This book is targeted both at the basic and
clinical scientists, in industry or academia, who wish a broad overview of the field and an
understanding of the data available from studies in humans. It will also be valuable for
physicians who want to understand the role that endothelial dysfunction may play in the
patients under their care, and the emerging opportunities for new treatments.
Sir John Vane

CONTRIBUTORS
Bagnall, A.J. Cooke, J.P.

Centre for Genome Research Division of Cardiovascular Medicine
Roger Land Building Stanford University School of Medicine
University of Edinburgh 300 Pasteur Drive
West Mains Road Stanford, CA 94305 5406
Edinburgh, EH9 3JQ USA
UK
Corti, R.
Bhagat, K. Division of Cardiology
Centre for Clinical Pharmacology University Hospital Zurich
The Rayne Institute CH-8091 Zurich
University College London Switzerland
5 University Street
London, WC1E 6JJ Creager, M.A.
UK Vascular Medicine and Atherosclerosis Unit
Brigham and Women’s Hospital and
Binggeli, C. Harvard Medical School
Division of Cardiology Boston, MA 02115
University Hospital Zurich USA
CH-8091 Zurich
Switzerland Deanfield, J.E.
Cardio-Thoracic Unit
Celermajer, D.S. Great Ormond Hospital for Children
Department of Cardiology NHS Trust
Royal Prince Alfred Hospital Great Ormond Street
Missenden Road London, WC1 3JH
Camperdown UK
Sydney, NSW 2050
Australia Drexler, H.
Department of Cardiology
Chowienczyk, P.J. Medical University Hannover
Department of Clinical Pharmacology Carl-Neuberg-Str. 1
St Thomas’ Hospital, UMDS D-30625 Hannover
Lambeth Palace Road Germany
London, SEI 7EH
UK
xi
xii Contributors
Evans, T.W. Mann, G.E.

Department of Critical Care Medicine Vascular Biology Research Centre
National Heart and Lung Institute School of Biomedical Sciences
Dovehouse Street King’s College London
London, SW3 6LY Campden Hill Road
UK London, W8 7AH
UK
Félétou, M.
Institut de Recherche Internationales Nava, E.
Servier Division of Cardiology
6 place des Pléiades University Hospital Zurich
92415 Courbevoie Cedex CH-8091 Zurich
France Switzerland
Haynes, W.B. Noll, G.

Department of Internal Medicine Division of Cardiology
University of Iowa University Hospital Zurich
Iowa City, IA 52241 CH-8091 Zurich
USA Switzerland
Hingorani, A.D. Poston, L.

Centre for Clinical Pharmacology Department of Obstetrics and Gynaecology
The Wolfson Institute for Biomedical St Thomas’ Hospital, UMDS
Research Lambeth Palace Road
University College London Medical School London, SE1 7EH
140 Tottenham Court Road UK
London, W1P 9LN
UK Radomski, A.S.
Department of Pharmacology
Kiowski, W. University of Alberta
Division of Cardiology Edmonton
University Hospital Zurich Alberta, T6G 2H7
CH-8091 Zurich Canada
Switzerland
Radomski, M.W.
Lüscher, T.F. Department of Pharmacology
Division of Cardiology University of Alberta
University Hospital Zurich Edmonton
CH-8091 Zurich Alberta, T6G 2H7
Switzerland Canada
MacAllister, R.J. Ritter, J.M.

Centre for Clinical Pharmacology Department of Clinical Pharmacology
The Wolfson Institute for Biomedical St. Thomas’ Hospital, UMDS
Research Lambeth Palace Road
University College London Medical School London, SE1 7EH
140 Tottenham Court Road UK
London, W1P 9LN
UK
Contributors xiii
Sudano, I. Vanhoutte, P.M.

Division of Cardiology Institut de Recherche Internationales Servier
University Hospital Zurich 6 place des Pléiades
CH-8091 Zurich 92415 Courbevoie, Cedex
Switzerland France
Taddei, S. Webb, D.J.

Department of Internal Medicine Clinical Pharmacology Unit and Research
University of Pisa Centre
Via Roma 67 University of Edinburgh
56100 Pisa Western General Hospital
Italy Crewe Road
Edinburgh, EH4 2XU
Vallance, P.J.T. UK
Centre for Clinical Pharmacology
The Wolfson Institute for Biomedical Williams, D.J.
Research Department of Obstetric and Gynaecology
University College London Medical School Imperial College School of Medicine
140 Tottenham Court Road Chelsea and Westminster Hospital
London, W1P 9LN Fulham Rd
UK London, SW10 9NH
UK
ACKNOWLEDGEMENTS
None of this would have been possible without the hard work and careful cross checking
done by Ann Wemyss. She also managed to keep us all to a time scale in a diplomatic and
persuasive manner. In addition we would like to thank Mark Miller for providing the overview
figures for each chapter.
xv
The gaseous mediator nitric oxide (NO) is synthesised in the vascular endothelium from the substrate L-arginine
in an enzymatic reaction catalysed by endothelial nitric oxide synthase. Nitric oxide influences the function of
circulating cells and the underlying smooth muscle to contribute to the tonic atheroprotective, thromboresistant
and vasodilator influence of the endothelium. The endothelial output of nitric oxide can be modulated over the
short-and long-term by both chemical and physical signals and might, in part, be genetically-determined.
Dysfunction of the endothelial:NO pathway may contribute to a variety of cardiovascular disorders including
essential hypertension, atherosclerosis and pre-eclampsia.
1 Endothelial Nitric Oxide
Aroon D.Hingorani and Patrick J.Vallance
Centre for Clinical Pharmacology, University College London, 140 Tottenham Court
Road, London, W1P 9LN, UK
Tel/Fax: 0171 209 6351; E-mail: a.hingorani@ucl.ac.uk
INTRODUCTION
In 1980 Furchgott and Zawadzki reported the observation that vascular relaxation induced
by acetylcholine was dependent on the presence of the endothelial lining of the blood vessel
(Furchgott and Zawadzki, 1980) If the endothelium was removed, the relaxation to
acetylcholine was abolished whereas the contractile response to noradrenaline and the dilator
response to glyceryl trinitrate were both preserved. Endothelium-dependent relaxation to
acetylcholine was shown to be mediated by the release of an unstable, diffusible factor
(endothelium-derived relaxant factor; EDRF) which had a biological half-life of a few
seconds (Furchgott, 1984; Furchgott, 1990) Subsequently the dilator responses to a variety
of other agonists including bradykinin, substance P, adenosine diphosphate, 5-
hydroxytryptamine, ß-agonists and certain neuropeptides were also shown to be endothelium-
dependent and mediated by EDRF (Moncada et al., 1991). The release of EDRF was also
shown to attenuate the response to certain endogenous vasoconstrictors (Martin et al., 1986).
For 6 years the nature of EDRF remained speculative until Furchgott and Ignarro
suggested independently that it might be a simple inorganic molecule—nitric oxide (NO).
This hypothesis was confirmed a year later when Palmer and colleagues and Ignarro showed
that vascular endothelial cells synthesise NO and that the known biological effects of EDRF
could be reproduced by exogenous administration of NO (Palmer et al., 1987; Ignarro et
al., 1987; Radomski et al., 1987a) It is now known that NO is synthesised from L-arginine
(Palmer et al., 1988a) in a reaction which utilises molecular oxygen, to generate NO and
the co-product L-citrulline (Palmer and Moncada, 1989).
The enzymes responsible for catalysing the conversion of L-arginine to NO (the nitric
oxide synthases) and their respective genes have now been identified, the biochemistry
of the L-arginine:NO pathway is well established and a variety of drugs are available
which modulate the activity of the system. In this chapter we discuss the physiological
roles of NO in the endothelium of the human cardiovascular system, describe how it is
synthesised and how this synthesis is regulated, and discuss some of the approaches used
to study NO biology in humans.
ENDOTHELIUM-DEPENDENT RELAXATION
The experiments of Furchgott and Zawadzki have been replicated in a wide variety of
human blood vessels in vitro and with a number of agonists. Endothelium-dependent
relaxation has been demonstrated in human conduit arteries (O’Neil et al., 1991; Chester
3
4 A.D.Hingorani and P.Vallance
et al., 1990; Thom et al., 1987; Greenberg et al., 1987; Toda and Okamura, 1989;
Kanamaru et al., 1989; Schoeffter et al., 1988; Luscher and Vanhoutte, 1988; Luscher et
al., 1987), small resistance vessels (Woolfson and Poston, 1990; Vila et al., 1991), and
veins (Thom et al., 1987; Collier and Vallance, 1990). However, it is clear that the response
to agonists varies between vessel types. For example, whilst acetylcholine causes near
maximal relaxation of human conduit arteries (Jovanovic et al., 1994; Thom et al., 1987;
Schoeffter et al., 1988; Yasue et al., 1990; Yang et al., 1991; Collins et al., 1993) and
resistance vessels (Cockcroft et al., 1994a; Chowienczyk et al., 1993; Imaizumi et al.,
1992; Linder et al., 1990), human saphenous veins (Yang et al., 1991; Lawrie et al.,
1990; Luscher et al., 1990; Thom et al., 1987), hand veins (Collier and Vallance, 1990;
Vallance et al., 1989a) and omental venules (Wallerstedt and Bodelsson, 1997) show
only a small relaxant response. Furthermore, studies in human and animal vessels show
that the response to acetylcholine is complex and comprises several distinct components:
endothelium-dependent relaxation (Bruning et al., 1994), endothelium-independent
contraction (a direct effect on muscarinic receptors on smooth muscle) (Vallance et al.,
1989b; Penny et al., 1995), indirect neurogenic relaxation through stimulation of
perivascular nerves (Loke et al., 1994) and, in certain specialised vessels, endothelium-
independent relaxation (Brayden and Large, 1986). The relative contribution of each
component of the response may vary between vessel types, between vascular beds and
between disease states. Therefore, in some instances, the overall response to acetylcholine
may not represent an accurate assessment of endothelial dilator function.
Studies in humans, in vivo and in vitro, confirm that acetylcholine causes vasodilatation
of conduit arteries (O’Neil et al., 1991; Chester et al., 1990; Thom et al., 1987; Greenberg
et al., 1987; Toda and Okamura, 1989; Kanamaru et al., 1989; Schoeffter et al., 1988;
Luscher and Vanhoutte, 1988; Luscher et al., 1987), resistance vessels vessels (Woolfson
and Poston, 1990; Hardebo et al., 1987; Vila et al., 1991), and veins (Thom et al., 1987;
Collier and Vallance, 1990), but again differences between vessels are apparent. In the
resistance beds, infusion of acetylcholine causes a graded vasodilatation and no constrictor
element has been reported (Vallance et al., 1989b; Cockcroft et al., 1994b; Chowienczyk et
al., 1993; Imaizumi et al., 1992; Linder et al., 1990), whereas in the coronary artery and
superficial veins acetylcholine causes a biphasic response with low doses causing
vasodilatation and higher doses causing constriction (Collier and Vallance, 1990; Angus et
al., 1991) (Figure 1.1). It is assumed that the dilator component is largely
endotheliumdependent whereas the constriction is mediated either by a direct action on
smooth muscle or by indirect neurogenic mechanisms. In superficial veins it has been
demonstrated directly that the dilator component of the response to acetylcholine is
endothelium-dependent in vivo (Collier and Vallance, 1990).
NITRIC-OXIDE MEDIATED VASODILATION
The generation of NO from L-arginine can be inhibited competitively by certain guanidino-

substituted analogues of arginine of which NG monomethyl-L-arginine (L-NMMA) has
been the most widely used (Figure 1.2).
Endothelial Nitric Oxide 5
Figure 1.1 Biphasic response to acetylcholine in human hand vein in vivo. Acetylcholine was infused into
hand veins partially preconstricted with noradrenaline. At low doses, acetylcholine produced vasodilatation, whereas
at higher doses it caused vasoconstriction. In some vessels endothelium was removed by infusions of distilled
water. In these vessels, the dilator effects of acetylcholine were lost and the constrictor effects enhanced.
Human Vessels In Vitro

L-NMMA inhibits the response to “endothelium-dependent” agonists in human vessels
in vitro (Wallerstedt and Bodelsson, 1997; Raddino et al., 1997; Jovanovic et al., 1994;
Vila et al., 1991; Crawley et al., 1990), and blocks the generation of NO by endothelial
cells maintained in culture (Palmer et al., 1988b). Its effects are competitive (being
overcome by excess L-arginine), stereospecific (the D-enantiomer is without effect)
(Radomski et al., 1990; Vallance et al., 1989a; Crawley et al., 1990; Jovanovic et al,
1994; Mollace et al., 1991) and selective (there is no evidence that L-NMMA affects
any other enzyme system, ion channel or receptor, in the µM concentration range used
[for review see (Vallance, 1996)]). Thus L-NMMA has proved a useful tool to confirm
that endothelium-dependent relaxation of human vessels is dependent upon generation
of NO. However, the ability of L-NMMA to block the relaxant response to acetylcholine
varies from vessel to vessel. For example, L-NMMA completely blocks acetylcholine-
induced relaxation in saphenous vein and internal mammary artery but in small resistance
arteries a substantial part of the relaxation persists even in the presence of a maximally
effective concentration of L-NMMA [see Chapter 4]. Endothelium-dependent but L-
NMMA-resistant relaxation has been seen in resistance vessels from animals and man
(Urakami Harasawa et al., 1997) and it seems likely that a considerable proportion of
Figure 1.2 Structures of arginine (substrate for nitric oxide synthesis) and two inhibitors, asymmetric
dimethylarginine (ADMA) and NG-monomethyl-L-arginine (L-NMMA). L-NMMA and ADMA are both
naturally-occuring compounds that compete with arginine and inhibit nitric oxide synthesis. Another endogenous
compound, symmetric dimethylarginine (SDMA), in which methyl groups are present on each of the guanidino
nitrogen atoms, does not act as an inhibitor of nitric oxide synthesis.
the response to acetylcholine is not mediated by endothelial generation of NO in resistance

vessels (Figure 1.3).
Studies In Vivo
In studies of the resistance vasculature of the human forearm in vivo, L-NMMA inhibits
the relaxation to acetylcholine (Vallance et al., 1989b; Chowienczyk et al., 1993),
bradykinin (Cockcroft et al., 1994b), vasopressin (Tagawa et al., 1993), substance P
(Tagawa et al., 1997), and 5-hydroxytryptamine (Bruning et al., 1993). L-NMMA also
inhibits relaxation to ACh in conduit arteries. In superficial hand veins, L-NMMA, but
not D-NMMA, blocks completely the relaxation to acetylcholine or bradykinin (Vallance
Figure 1.3 Diverse effects of acetylcholine on vascular tone. Acetylcholine may cause vascular contraction or
relaxation by endothelium-dependent or endothelium-independent mechanisms. Endothelium dependent relaxation
is mediated via the release of nitric oxide or endothelium-derived hyperpolarising factor (EDHF). Endothelium-
independent contraction is induced by direct activation of muscarinic cholinoceptors on the vascular smooth
muscle cell. In certain specialised vessels (e.g. the lingual artery) acetylcholine may produce endothelium-
independent relaxation.
et al., 1989a), and inhibits the generation of NO as detected by a porphyrinic microsensor

(Vallance et al., 1995). These effects of L-NMMA are reversed by L-arginine but not D-
arginine, confirming the specificity of the responses seen. The dilator response to
acetylcholine in the coronary artery in vivo is also nearly completely abolished by L-
NMMA (Quyyumi et al., 1995b; Lefroy et al., 1993). However, similar to the situation in
vitro, the dilator response to acetylcholine in resistance vessels is only partly inhibited by
L-NMMA (Vallance et al., 1989b; Quyyumi et al., 1995a; Lefroy et al., 1993). In the
forearm resistance bed, infusion of acetylcholine into the brachial artery causes a large
increase in blood flow which can be inhibited by 40–50% by L-NMMA (Vallance et al.,
1989b). Again this result is similar to observations in animals and is consistent with the
idea that acetylcholine causes vasodilatation through multiple mechanisms in resistance
vessels.
The vascular endothelium has receptors for agonists but can also respond to physical
stimuli (Davies, 1995; Lansman, 1988). Ion channels that respond to stretch (Lansman
et al., 1987) and flow (Olesen et al., 1988; Siegel et al., 1996) have been identified and
the endothelium responds to increased shear stress by increasing the generation of NO
(Meredith et al., 1996; Kanai et al., 1995; Buga et al., 1991; Lamontagne et al., 1992;
Noris et al., 1995). This is responsible for the phenomenon of flow-mediated
vasodilatation (Meredith et al., 1996; Lieberman et al., 1996; Kuo et al., 1990; Pohl et
Figure 1.4 Basal nitric oxide generation in the human forearm. L-NMMA produces a dose-dependent fall in
resting forearm blood flow. L-NMMA (1, 2 and 4mol/min) was infused into the the brachial artery of one arm and
blood flow measured in both arms. Local inhibition of nitric oxide synthesis leads to a substantial fall in resting
flow and an increase in peripheral resistance in the infused arm (open triangles), with no change in flow in the
control arm (closed boxes).
al., 1986; Celermajer et al., 1992), which might be a mechanism for maintaining constant
shear stress within the vascularure [see Chapter 10 and (MacAllister and Vallance, 1996)].
Increased flow through the brachial or coronary arteries in humans is associated with
vasodilatation of the vessel and this response is abolished by L-NMMA (Meredith et al.,
1996; see Chapter 10).
Basal Generation Of Nitric Oxide

In addition to its effects on agonist or shear stress-stimulated dilatation, L-NMMA affects
resting vessel tone (Yang et al., 1991). The largest effect is seen in resistance vessels (Vallance
et al., 1989b), with human conduit arteries and veins showing little or no increase in basal
tone in response to L-NMMA. Vascular resistance nearly doubles when L-NMMA is infused
indicating that basal generation of NO provides a major continuous vasodilator influence
in the human arterial circulation (Figure 1.4) (Vallance et al., 1989b). This effect has been
demonstrated in the forearm arterial bed and in the coronary (Lefroy et al., 1993), renal
(Haynes et al., 1997; Dijkhorst Oei and Koomans, 1998) and cerebral (White et al., 1998)
circulations of healthy volunteers. Systemic infusion of L-NMMA causes a substantial

increase in systemic vascular resistance and elevates blood pressure (Haynes et al., 1993;
Stamler et al., 1994).
The mechanisms underlying basal generation of NO remain obscure. It is possible that
the increased shear stress on the arterial side of the circulation provides a continuous stimulus
for NO generation. However, this would not explain why basal NO generation is seen
predominantly in resistance rather than conduit vessels, nor why the basal NO generation
persists when arterial vessels are studied in vitro under conditions of no flow. The
physiological function of basal NO release is also unclear. At the most simplistic level,
basal generation of NO might provide a natural counterbalance against the constrictor action
of the sympathetic nervous system or the basal generation of endothelin [see Chapter 2].
Like noradrenaline, NO is a rapidly acting mediator and perhaps the two act in concert to
allow for central (sympathetic nervous system) and local (endothelial) regulation of vascular
tone. Basal, or shear stress activated NO may also contribute to vascular compliance or
allow the cardiovascular system to adapt readily to changes in intravascular volume [for
discussion see (MacAllister and Vallance, 1996)].
Other Actions Of Endothelial Nitric Oxide

Although vasodilatation is the most obvious and well studied effect of endothelial NO,
many other actions have been identified in studies in animals and in vitro. Nitric oxide
inhibits platelet aggregation (Radomski et al., 1990), prevents adhesion of platelets to
the endothelial surface (Radomski et al., 1987b) and induces disaggregation of aggregating
platelets [see Chapter 5]. In addition, it inhibits the activation and expression of certain
adhesion molecules (Takahashi et al., 1996; Khan et al., 1996; Biffl et al., 1996; De
Caterina et al., 1995) and modulates adhesion of white cells (Bath et al., 1991). Smooth
muscle cell replication (Garg and Hassid, 1989; Nakaki et al., 1990) and migration (Sarkar
et al., 1996) is inhibited by NO and, in experimental models, an intact endothelial NO
system is important to limit the degree of intimal hyperplasia that occurs after balloon
injury (Lee et al., 1996; Wolf et al., 1995), or the neo-intima formation that occurs during
cholesterol feeding (Naruse et al., 1994; Cayatte et al., 1994). In vivo transfer of the
eNOS gene into balloon injured rat carotid artery restores NO production in this vessel to
normal levels and and inhibits neointimal hyperplasia (Janssens et al., 1998; von der
Leyen et al., 1995). Whilst many of these effects have also been observed using human
cells in vitro there are few data on human blood vessels studied in vitro or in vivo. Infusion
of L-NMMA is associated with a decrease in bleeding time but no effects on platelet
aggregation have been detected (Simon et al., 1995; Remuzzi et al., 1990). However,
inhaled NO (100–884 ppm) has been shown to increase bleeding time and reduce agonist-
stimulated platelet aggregation (Gries et al., 1998)
MEASUREMENT OF NITRIC OXIDE IN HUMANS
Nitric oxide has a biological half life of a few seconds in biological solution (Cocks et
al., 1985; Griffith et al., 1984) and rapidly degrades in blood to nitrite and thence to
nitrate, both of which are largely inactive. Although circulating adducts of NO have been
identified (Stamler et al., 1992b; Stamler et al., 1992a; Keaney, Jr. et al., 1993), most
data suggest that endothelial NO acts close to its point of synthesis and does not have
significant downstream or circulating effects. Because of its short half-life, biochemical
assessment of NO generation has proved difficult. Free NO can be measured using a
porphyrinic microsensor (Malinski et al., 1993b; Malinski et al., 1993a; Malinski and
Taha, 1992; Vallance et al., 1995). Although useful for detecting the kinetics of NO release,
it cannot be used as a quantitative measure of NO production because of the instability of
the molecule (Baylis and Vallance, 1998). Nitrite is unstable in blood and its concentration
in plasma provides only a glimpse of NO in an intermediary state of metabolism. Nitrate
is a stable end product of NO and its concentration can readily be assessed in plasma and
urine. However, a substantial proportion of the circulating nitrate derives from the diet
and the remainder may come from NO-generating cells other than the vascular endothelium
(Baylis and Vallance, 1998). An alternative method to quantify NO production involves
measuring the conversion of exogenously administered 15N-labelled arginine to 15N-
nitrate—an approach which can be used in vivo (Hibbs, Jr. et al., 1992; Forte et al., 1997;
Macallan et al., 1997). Although this has the advantage that all of the measured nitrate
derives from the L-arginine:NO pathway it still does not allow precise identification of
the cellular source of the NO, nor does it indicate how much of the NO generated was
biologically active.
Overall, measurement of NO metabolites has confirmed the existence of NO generation
in humans but each of the methods used has significant drawbacks when assessing the
activity of endothelial NO in health or disease. A full critique of biochemical assessment of
NO in humans has been published elsewhere (Baylis and Vallance, 1998) and is outside the
scope of the present chapter.
NITRIC OXIDE SYNTHASE IN THE VASCULAR ENDOTHELIUM
The biosynthesis of NO from L-arginine and molecular oxygen is mediated by a family of

three enzymes—the nitric oxide synthases (NOSs) (Figure 1.5) (Forstermann et al., 1994b;
Forstermann et al., 1994a; Forstermann et al., 1993; Knowles and Moncada, 1994).
Endothelial NOS (eNOS), like neuronal NOS (nNOS), was originally characterised by: (1)
a constitutive activity responsible for generating low levels of NO in the basal state; (2)
regulation by intracellular calcium concentration (Mulsch et al., 1989; Mayer et al., 1989);
and (3) a restricted pattern of expression which not only includes the endothelium, but also
platelets (Sase and Michel, 1995), placenta (Eis et al., 1995; Myatt et al., 1993), myocytes
(Feron et al., 1996; Wei et al., 1996) and bronchiolar epithelium (Shaul et al., 1994). This
contrasts with the inducible isoform of NOS (iNOS) which requires an inflammatory stimulus
for its expression (Hibbs, Jr. et al., 1988; Marletta et al., 1988; de Vera et al., 1996), can be
induced by cytokines in a variety of cell types including macrophages (Ravalli et al., 1998;
Nicholson et al., 1996), vascular smooth muscle cells (Ravalli et al., 1998; Kolyada et al.,
1996), endothelial cells, neutrophils (Evans et al., 1996; Wheeler et al., 1997) and eosinophils
(del Pozo et al., 1997), appears functionally independent of calcium (McCall et al., 1991),
and synthesises NO at relatively high rates. However, this classification of NOS isoforms
according to inducibility and calcium-dependence is now considered over-simplistic since
eNOS is subject to transcriptional regulation (Harrison et al., 1996; Wang and Marsden,
1995a), and the apparent calcium-independence of iNOS is, in fact, a manifestation of the
Figure 1.5 Human nitric oxide synthase gene family. Endothelial nitric oxide synthase (eNOS) is one of a
family of three isoforms, the other two isoforms being neuronal (nNOS) and inducible (iNOS). Each isoform is a
product of a separate gene but all are highly homologous particularly in regions encoding the highly conserved
binding sites for calmodulin and enzyme co-factors. The location of these binding sites is illustrated in the upper
panel.
permanent, tight binding of a Ca2+/calmodulin complex to this isoform of the enzyme (Cho et
al., 1992).
Enzymatic Synthesis Of Endothelial Nitric Oxide

In common with the other isoforms, endothelial nitric oxide synthase has a C-terminal
reductase domain (which bears much sequence and functional homology to the cytochrome
P450-like haemproteins) and anN-terminal oxidative domain (Wang andMarsden, 1995a).
Whilst enzyme monomers can mediate electron exchange, the active form of one eNOS
enzyme is a homodimer (Rodriguez Crespo et al., 1996; Venema et al., 1997) catalysing
the formation of NO from the amino-acid L-arginine and molecular oxygen in a reaction
which involves the five electron oxidation of the terminal guanidino nitrogen of arginine.
NADPH is a co-substrate for this reaction, which also involves the co-factors FAD, FMN,
enzyme-bound haem and tetrahydrobiopterin. L-arginine is oxidised through an intermediate
(NG-hydroxyl-L-arginie) to L-citrulline—a co-product formed in equimolar quantities with
NO. Binding sites for FAD, FMN, calmodulin and NADPH (apparent from consensus
sequence motifs in the primary structure of the protein) are located in the C-terminal
reductase domain, and are encoded by distinct exons or groups of exons. The binding site
for haem/arginine is less well documented but is thought to reside in the N-terminal oxidative
end of the protein.
Gene Localisation, Structure And Polymorphism

Each NOS isoform is the product of a separate gene and the structure and chromosomal
location of each of these has been ascertained in man (Wang and Marsden, 1995b) (Figure
10.5). At the DNA level, there is a 50–60% sequence homology between isoforms within a
species and this is particularly prominent in the regions encoding peptide domains known
to subserve a particular biological function (e.g. co-factor binding). Between-species
homology for a specific isoform is much greater, with the bovine, murine and human eNOS
gene sequences exhibiting 80–90% homology. Despite this evolutionary conservation, human
eNOS has been shown to exhibit significant inter-individual sequence variation
(polymorphism) (Nadaud et al., 1994; Marsden et al., 1993; Hingorani et al., 1997;
Miyamoto et al., 1998; Markus et al., 1998; Wang et al., 1996). A variety of both bi- and
multi-allelic polymorphisms have been identified in the promoter and introns of the gene
(Figure 1.6). A polymorphism in intron 4 of the gene may be a marker for coronary artery
disease and, may also be a marker for (or directly influence) the amount of NO generated
by the enzyme (Wang et al., 1997). Thus far, only one genetic variant affecting the aminoacid
sequence has been identified. A G→T substitution in exon 7 of the gene predicts a glutamic
acid (Glu)→spartic acid (Asp) substitution at residue 298 of the mature protein (Hingorani
et al., 1995; Yasue et al., 1995). Whether any of these sequence variants alters transcriptional
regulation or activity of the encoded protein is not yet known, but the Glu298Asp
polymorphism, which has been identified in both Caucasian (Hingorani et al., 1995) and
Japanese subjects (Yasue et al., 1995) (albeit at different allele frequency), has been
associated with a variety of cardiovascular disorders including essential hypertension in
the Japanese (Miyamoto et al., 1998), coronary artery spasm (Yasue et al., 1995; Yoshimura
et al., 1997), coronary atherosclerosis (Hingorani et al., 1997), myocardial infarction
(Shimasaki et al., 1998; Hibi et al., 1998) and the NOS 3 gene has also been linked to pre-
eclampsia (Arngrimsson et al., 1998). Comparison with the recently solved crystal structure
of murine inducible nitric oxide synthase (iNOS) (Crane et al., 1997), suggests that residue
298 of human eNOS (homologous with residue 308 of murine iNOS) is located in a loop
between two ß-pleated sheets on the external surface of the protein and is proximal to
residues critical for substrate binding e.g. residue Glu 371 in murine iNOS (homologous to
Glu 361 in human eNOS).
Expression And Subcellular Localisation

Originally cloned and isolated from vascular endothelium, eNOS is now known to be
expressed in a variety of other cell types including platelets (Sase and Michel, 1995),
cardiac myocytes (Feron et al., 1996; Wei et al., 1996), the syncytiotrophoblast of the
placenta (Buttery et al., 1994) and the hippocampus of the brain (Dinerman et al.,
1994). Endothelial NOS is highly membrane-bound (Busconi and Michel, 1993). Recent
studies have shown that eNOS can be found in the Golgi apparatus (Sessa et al., 1995)
and in small protein-rich invaginations of the plasma membrane called caveolae (Shaul
et al., 1996). Within the caveolae of endothelial cells, eNOS is bound to a protein called
caveolin-1—this interaction involves a conserved 20 amino acid region within
Figure 1.6 Polymorphisms of the human endothelial nitric oxide synthase gene (NOS 3). Panel A illustrates
the gene structure intron/exon arrangement and polymorphisms of the human NOS 3 gene. Single nucleotide bi-
allelic polymorphisms are shown above the gene. The variable number tandem repeat (VNTR) polymorphism in
intron 4 is also bi-allelic with individuals having 4 or 5 repeats of a 27bp sequence element. The multi-allelic CA
repeat polymorphism in intron 13 is highly polymorphic with allele sizes ranging from 18 to 36 CA repeats
(Hingorani, 1997).
caveolin-1 and a proposed caveolin-binding sequence in eNOS in the oxidative domain

(Michel et al., 1997b; Feron et al., 1996). Additional membrane binding interactions also
exist. The presence of glycine as the second amino-acid residue found in eNOS, serves as
an acceptor site for myristic acid which is critical for the binding of eNOS to the plasma
membrane (Busconi and Michel, 1994; Busconi and Michel, 1993). Artificial site-directed
mutagenesis of this residue converts eNOS from a membrane-bound to a cytosolic enzyme
(Busconi and Michel, 1993). Palmitoylation of eNOS at two cysteine residues near the N-
terminus stabilises this membrane binding (Robinson and Michel, 1995; Robinson et al.,
1995). This dual acylation is unique among the NOS isoforms (Michel and Feron,
1997). The functional significance of these processes which affect NOS localisation within
the cell is discussed below.
Regulation Of Expression And Activity

The NOS isoforms are regulated at many levels from gene transcription to posttranslational
modification (Wang and Marsden, 1995a). Sequence analysis of the promoter regions of
each enzyme isoform reveals potential sequences for regulation through a variety of
transcription factors including, including those induced by TGF-ß (NF-1), cAMP (AP-2)
and phorbol esters (AP-1) (Wang and Marsden, 1995a). Transcription of the eNOS gene is
upregulated by increased shear stress at the endothelial cell surface (Topper et al., 1996;
Uematsu et al., 1995; Ranjan et al., 1995), by TGF-ß (Inoue et al., 1995) and by oestrogens
(Armour and Ralston, 1998; MacRitchie et al., 1997; Kleinert et al., 1998) but the cellular
mechanisms and transcription factors mediating these effects have yet to be completely
defined. The eNOS gene promoter contains a single shear stress response element (SSRE)
(Marsden et al., 1993; Nadaud et al., 1994; Miyahara et al., 1994), comprising a 6 base
pair sequence motif originally identified as transducing the shear-induced transcriptional
activation of the PDGF-gene and subsequently identified in a variety of other endothelially
expressed shear-responsive genes. Whether this sequence underlies shear-induced
transcription of eNOS has yet to be formally confirmed. Although the eNOS promoter does
not contain the full 22 base pair oestrogen response element (ERE)—a sequence motif
mediating the transcriptional response to the oestrogen/oestrogen receptor complex—it does
contain a number of so-called “half-sites” comprising half of the full-length ERE consensus
sequence which in other genes have been shown to act in concert to mediate a transcriptional
response to oestrogen (Miyahara et al., 1994). Again it is not clear whether it is these
sequences which confer oestrogen responsiveness (Kleinert et al., 1998). Transcriptional
regulation of the eNOS gene by these two stimuli may underlie the increase in NO generation
seen physiologically following exercise training (Wilson and Kapoor, 1993) and in pregnancy
(Williams et al., 1997).
Post-translational Modification of eNOS

The endothelial NOS is also known to undergo post-translational modification, and this
serves as a further potential mechanism for regulating activity. As well as N-terminal
myristoylation (Busconi and Michel, 1994; Busconi and Michel, 1993) and palmitoylation
(Robinson and Michel, 1995; Robinson et al., 1995) which determine subcellular targeting,
the enzyme also undergoes phosphorylation (Michel et al., 1993; Corson et al., 1996).
Although myristoylation appears irreversible, palmitoylation/depalmitoylation is a
reversible process thought to be subject to regulation (Robinson et al., 1995). Bradykinin,
which stimulates NO generation by the endothelium has been shown to modulate this
process. Endothelial NOS has the potential to be phosphorylated at both tyrosine (Garcia
Cardena et al., 1996) and serine (Corson et al., 1996; Michel et al., 1993) residues. In
endothelial cells, phosphorylation of eNOS at serine residues occurs following exposure
to agonists and shear stress (Corson et al., 1996; Michel et al., 1993) and in some cases
has been associated with subcellular translocation of the protein. Fluid shear stress may
induce tyrosine phosphorylation and activation of eNOS independent of Ca2+/calmodulin
(Fleming et al., 1997).
Figure 1.7 Intracellular localisation and activation of eNOS. Active eNOS is a homodimer. Trafficking of the
enzyme to subcellular membrane compartments such as the Golgi apparatus and caveolae appears to occur. Caveolae
may be sites where arginine transporters and receptors are co-localised in close proximity to membrane bound
eNOS. Caveolins are negative allosteric regulators of the enzyme. Caveolin-mediated inhibition of enzyme activity
may be removed by activation of calcium-calmodulin by certain agonists e.g. bradykinin. In some cases, agonist-
mediated activation is accompanied by enzyme translocation from membrane to cytosol.
Mechanism of Agonist-induced eNOS Activation

The increase in activity of pre-formed eNOS mediated by exposure of endothelial cells to
endothelium-dependent agonists has been shown to be dependent on an increased
concentration of intracellular calcium and is mediated by the binding to eNOS of the calcium-
activated protein calmodulin (Marletta, 1993). This binding and activation appears to be
associated with the displacement of caveolin from the eNOS/caveolin protein complex
(Michel et al., 1997a). It has been proposed that caveolin and calmodulin act as opposing
modulators of eNOS activity, the former acting as an allosteric inhibitor and the latter as an
allosteric activator (Michel and Feron, 1997; Michel et al., 1997b). The concentration of
eNOS within caveolae has additional potential implications for regulation of enzyme activity.
Caveolae may serve as regions of the plasma membrane where there is a high concentration
of mechanosensing molecules (e.g. shear stress sensors), molecules which mediate signal
transduction, and arginine transporters (McDonald et al., 1997). A microenvironment within
caveolae might also modulate eNOS activity by controlling the local concentration of co-
factors and substrate. Clearly this has potential implications for changes occuring in disease.
Whilst overall expression of eNOS may be unchanged in disease, enzyme activity might be
drastically altered by changes in the subcellular localisation of eNOS or the proteins with
which it interacts. (Figure 1.7)
Substrate Concentration-Enzyme Activity Relationship
L-arginine circulates in plasma at a concentration of 50–100 µM and enters cells largely

but not exclusively through the cationic y+ amino acid transporter. Within the endothelial
cell concentrations of L-arginine of up to 800 µM have been measured (Baydoun et al.,
1990). These concentrations are many fold higher than the Km of the enzyme for L-arginine
of (1–4 µM) calculated from the activity of partially purified native or recombinant
enzymes (Charles et al., 1996; Bredt and Snyder, 1990). In principle, therefore, there
should be no situation in which the activity of the enzyme is substrate-limited. However,
several studies in humans in vivo have identified biological effects of L-arginine which
have been attributed to increased NO generation via the provision of excess substrate
(Bode Boger et al., 1994; Mehta et al., 1996; Imaizumi et al., 1992). In some instances
the reported effects seem unlikely to be due to this mechanism. For example, the
vasodilation and hypotension which occurs when very large doses of L-arginine are infused
(sufficient to increase the plasma concentration of arginine to 5–10 mM or higher), are
also seen with D-arginine and certain other amino acids which are not substrates for
NOS (Rhodes et al., 1996; MacAllister et al., 1995). However, particularly in certain
disease states such as atherosclerosis, the actions of arginine do appear to be stereospecific
and related to NO generation. The mechanism of this effect (termed the “arginine paradox”)
has not been determined but might relate to: (1) a disease-induced fall in substrate
concentration (at least in the microenvironment of the enzyme); (2) alteration of the Km
of the enzyme; (3) a deficiency of one or more co-factors; or (4) post-translational
modifications of the enzyme which affect the binding of substrates or co-factors, or the
sub-cellular localisation of the enzyme (Boger et al., 1996). Another possibility is that in
certain disease states there is an increase in the level of an endogenous enzyme inhibitor
which acts at the arginine binding site. The NOS inhibitor L-NMMA, which has been
widely used to probe the biology of the L-arginine:NO pathway and a closely related
compound, asymmetric dimethylarginine (ADMA), are endogenous naturally occuring
compounds.
Endogenous eNOS Inhibitors
Guanidino nitrogens in arginine residues present within proteins are methylated by the
action of protein methylase I. Upon hydrolysis of the proteins free L-NMMA and the
dimethylarginines ADMA and SDMA are released (Figures 1.2 and 1.8). These compounds
are synthesised by human endothelial cells (MacAllister et al., 1996a), circulate in plasma
and are excreted in urine (Vallance et al., 1992). L-NMMA and ADMA are both inhibitors
of NOS (Calver et al., 1993; MacAllister et al., 1994b) and all three methylated amino
acids can prevent arginine entry through the y+ transporter (MacAllister et al., 1994a). A
specific enzyme exists (dimethyl arginine dimethylamino hydrolase; DDAH) for
metabolising ADMA and L-NMMA and this has been identified in human endothelial cells
and blood vessels (MacAllister et al., 1996a). Increased circulating concentrations of ADMA
are found in certain disease states (Pickling et al., 1993; Vallance et al., 1992; MacAllister
et al., 1996b; Boger et al., 1997) and it is possible that local accumulation of this compound
contributes to impaired endothelial NO generation and competes with L-arginine thereby
rendering NOS arginine-sensitive in vivo. (Figure 1.8)
Figure 1.8 Synthesis and metabolism of ADMA. L-arginine is converted to nitric oxide by nitric oxide synthase.
However, it can also be converted to the inhibitor ADMA. The precise synthetic route is not clear but probably
involves methylation of arginine residues in proteins. Free ADMA is metabolised to citrulline by the action of
dimethylarginine dimethylaminohydrolase (DDAH). Citrulline is subsequently recycled to arginine.
MOLECULAR TARGETS FOR ENDOTHELIAL NITRIC OXIDE
Nitric oxide is non-polar, diffuses freely across cell membranes and therefore has a number
of potential fates depending on the target cell and chemical entity with which it interacts. It
is capable of binding to or reacting with a variety of chemical moieties including reactive
oxygen species (e.g. superoxide and hydroxyl radicals) and with metal-containing proteins
(e.g. soluble guanylate cyclase, cytochrome c oxidase).
Activation of Soluble Guanylate Cyclase

It is the binding and activation by NO of the haem-containing protein soluble guanylate
cyclase (sGC) that explains the elevation in cyclic guanosine monophosphate (cGMP)
observed in target cells following exposure to NO, and which accounts for many of its
biological effects (Ignarro, 1990a; Furchgott and Jothianandan, 1991). Soluble guanylate
cyclase is a heterodimeric protein containing distinct catalytic and haem-binding domains
(Hobbs, 1997). NO-induced activation of sGC is thought to require its binding to the haem
moiety to form a nitrosyl-haem complex. The conformational change induced in the
heterodimer by this binding is thought to expose the catalytic domain of sGC to its substrate
GTP, a process which leads to the formation of cGMP (Hobbs, 1997). In the vascular smooth
muscle cell (the prototypic target cell), there then follows an activation of cGMP-dependent
protein kinases and phosphorylation of intracellular proteins including myosin light chain
kinases and calcium-activated potassium channels which completes the NO-mediated
signalling cascade. Although it has been suggested that NO-mediated vasorelaxation can
occur in certain systems independently of an elevation in cGMP (Bolotina et al., 1994)
these findings require further confirmation and, in most vessels, activation of sGC seems to
account fully for the vasorelaxant effects of NO.
Interaction With Cytochrome C Oxidase

Cytochrome c oxidase (a key oxygen-binding protein in the mitochondrial electron
transport chain) also appears to be a cellular target of NO in a wide variety of tissues
studied (Brown, 1997; Borutaite and Brown, 1996; Brown, 1995; Brown and Cooper,
1994; Torres et al., 1997; Giuffre et al., 1996; Cleeter et al., 1994). NO binds with high
affinity to the oxygen binding site of the enzyme raising the apparent Km of cytochrome
oxidase for oxygen (Brown, 1997). It has been suggested that this renders mitochondrial
respiration sensitive to oxygen concentration over the normal physiological range thereby
allowing mitochondrial respiration to be regulated by physiological changes in oxygen
concentration. This area is of considerable potential physiological importance (for recent
review see (Torres and Wilson, 1996)).
Chemical Reactions of Nitric Oxide

NO released into the bloodstream and tissue fluids also has the potential to participate in a
variety of chemical reactions which would be expected to terminate the physiological activity
of free NO (Ignarro, 1990b). Potential reactions include combination with oxygen (to form
nitrite [NO2-]), with superoxide anion [O2-] (to generate peroxynitrite [ONOO-]) and with -
SH groups in peptides, proteins, amino acids and other small molecules to yield nitrosothiols
[R-SNO] (Ignarro, 1990b; Darley-Usmar et al., 1995). The reaction of NO with haemoglobin
leads rapidly to the formation of NO2- and methaemoglobin. Nitrite formed by the oxidation
of NO itself undergoes rapid oxidation to nitrate [NO3-] (a stable end-product). Under
physiological conditions it is likely that the activity of superoxide dismutase is sufficient to
prevent the the formation of significant amounts of ONOO- from the reaction between NO
and O2- (Beckmann and Koppenol, 1996). However, during inflammation (and other disease
states such as diabetes) when the production of both of these species is increased, there
may be significant generation of ONOO- and thence other damaging free radicals which
may contribute to the inflammatory process (Bhagat and Vallance, 1996).
The reversible interaction of NO with haemoglobin at its haem centre and to the
sulphydryl (-SH) group of a cysteine at residue 93 in the -globin chain (and the modulating
effect of molecular oxygen on this process) has been the subject of much recent interest
(Jia et al., 1996). The binding of NO to haemoglobin and its inactivation (see above)
would be expected to limit the immediate sphere of action of gaseous NO. However, the
recent observation that nitroso-Hb (a potential product of the NO/Hb interaction) might
itself act as a vasodilator by releasing NO at a distant site (Jia et al., 1996; Gow and
Stamler, 1998; Stamler et al., 1997) requires further verification. For the most part NO
can be considered a local rather than a circulating mediator. NO can also react with -SH
groups in other circulating substances including, homocysteine and albumin (Keaney et
al., 1993). The nitrosothiols which form have also been shown to behave as NO donors
in vitro and in vivo, but it is as yet unclear whether these substances play a major role in
normal circulatory physiology.
CONCLUSIONS
The importance of L-arginine:NO pathway in regulation of vascular tone, and systemic

blood pressure and in its contribution to the thromboresistant and atheroprotective functions
of the blood vessel wall have become clear over the last decade. The identification of this
signalling pathway has also fuelled the current interest in the biology of the endothelium as
a whole. There are, however, still a number of questions which await answers. What processes
are responsible for the basal release of NO from some vessel types but not others? How do
the mechanisms mediating physiological release of NO in response to physical stimuli (such
as flow) differ from those which subserve agonist-stimulated NO release? Which mechanisms
account for the “arginine paradox”?. It is hoped that the answers to these questions will be
forthcoming and that a better understanding of the mechanisms involved will enhance the
potential for the therapeutic manipulation of this important system.
Many of the chapters which follow discuss the evidence that NO-mediated dilation is
altered in disease states and that this might contribute to atherogenesis and other
cardiovascular disorders. It is increasingly clear that changes in NO-mediated effects may
also arise due to individual genetic differences in the activity or expression of eNOS,
posttranslational modification of the enzyme, endogenous inhibitors, and chemical reactions
of NO prior to its effect on target molecules. This book documents some of the functional
changes in the pathway seen in disease states.
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The endothelium plays a key role in the regulation of vascular tone, coagulation, lipid transport and immunological
reactivity. Endothelin-1, a member of a family of 21-amino acid peptides, is produced by the endothelium in
response to a number of stimuli, including exposure to epinephrine, angiotensin II and hypoxia. Three distinct
endothelin isoforms have been identified, termed endothelin-1, endothelin-2 and endothelin-3, of which endothelin-
1 is the most potent vasoconstrictor and of most importance functionally. Endothelin-1 produces slow onset and
sustained vasoconstriction through its actions on vascular smooth muscle cells. There are two distinct subtypes of
endothelin receptor, termed ETA and ETB, each the product of a separate gene. ETA receptors are located on
vascular smooth muscle cells and mediate vasoconstriction. The importance of endogenous endothelin-1, acting
via the ETA receptor, in the maintenance of basal vascular tone has been demonstrated through the use of selective
endothelin receptor antagonists in both local and systemic studies. ETB receptors are found on both endothelial
and vascular smooth muscle cells where their stimulation mediates vasodilatation and vasoconstriction respectively.
Local studies suggest the overall balance of ETB activation favours vasodilatation. In addition to its potent presser
effects, endothelin-1 exerts mitogenic effects on the cardiovascular system and is intimately linked with the
renin-angiotensin, L-arginine/nitric oxide and sympathetic nervous systems. Endothelin-1 has been implicated in
the pathogenesis of atherosclerosis, in the increase in peripheral resistance and the structural cardiac and vascular
changes seen in hypertension and chronic heart failure, and in a range of pathological conditions affecting the
lung, kidney, gut and central nervous system. Following promising results in animal studies, and early clinical
results in humans, large scale clinical trials are now underway in hypertension and heart failure to assess the
therapeutic potential of endothelin receptor antagonists in humans.
Key words: Endothelin, endothelin converting enzyme, human, cardiovascular, physiology, blood pressure.
2 The Endothelin System: Physiology
Alan J.Bagnall1 and David J.Webb2

1
Centre for Genome Research, Roger Land Building, The University of Edinburgh,
West Maino Road, Edingurgh, EH9 3JQ, UK
2
Clinical Pharmacology Unit and Research Centre, The University of Edinburgh,
Western General Hospital, Edinburgh EH4 2XU, Crewe Road, UK
INTRODUCTION
Endothelin-1 is the most potent vasoconstrictor and pressor agent currently identified
and was originally isolated and characterized by Yanagisawa and colleagues (1988) from
the culture media of aortic endothelial cells. Subsequently, two further isoforms, termed
endothelin-2 and endothelin-3, were identified along with structural homologues isolated
from the venom of Actractaspis engaddensis, known as the sarafotoxins. Each of the
mature isoforms consists of 21 amino acids linked by two constraining intra-chain
disulphide bonds. A highly conserved C-terminal sequence is mandatory for biological
function of the peptide (Figure 2.1). Although the isoforms are structurally similar,
endothelin-1 appears to be the predominant isoform involved in cardiovascular regulation
and is the only isoform produced constirutively by endothelial cells (Inoue et al., 1989).
The rapid development of selective endothelin receptor antagonists has led to an explosion
of research in this field. This work has demonstrated the therapeutic potential for
pharmacological manipulation of the endothelin system in a range of cardiovascular
conditions.
ENDOTHELIN GENERATION
The human genes for endothelin-1, endothelin-2 and endothelin-3 are located on
chromosomes 6, 1 and 20 respectively. Regulation of endothelin-1 synthesis is determined
primarily at the level of gene transcription via the influence of promoter regions located
upstream (5') of the preproendothelin-1 gene. Of these, a GATA binding site mediates
basal levels of gene transcription, whilst AP-1, nuclear factor and a hexonucleotide
sequence are thought to be regulated by angiotensin II, transforming growth factor ß and
acute phase reactants respectively. Further post-transcriptional modulation may occur
via selective destabilisation of preproendothelin-1 mRNA via ‘suicide motifs’ present in
the non-translated 3' region of this molecule. These may determine the short (15 minute)
half-life of preproendothelin-1 mRNA and thereby prevent excessive endothelin-1
production (Inoue et al., 1989). Factors known to promote endothelin-1 production include
thrombin, insulin, cyclosporine, epinephrine, angiotensin II, cortisol, inflammatory
mediators, hypoxia and vascular shear stress. Endothelin production is inhibited by nitric
oxide, nitric oxide donor drugs and dilator prostanoids via an increase in cellular cGMP,
and natriuretic peptides via an increase in cAMP levels (reviewed by Gray and Webb,
31
32 A.J.Bagnall and D.J.Webb
Figure 2.1 Structure of the endothelin family of peptides, and the related snake venom peptide sarafotoxin S6c.
Shaded amino acids indicate differences from endothelin-1.
The Endothelin System: Physiology 33
Figure 2.2 Factors that alter endothelin-1 (ET-1) synthesis and the pathway for endothelin-1 generation. IL-
1=interleukin-1; TGFß=transforming growth factor ß; LDL=low density lipoprotein; ANP, BNP, CNP =atrial,
brain and c-type natriuretic peptides. See text for details.
1995). The factors affecting endothelin generation and the synthetic pathways involved are
illustrated in Figure 2.2.
The mature endothelin-1 peptide is generated by enzymatic cleavage of the initial
preproendothelin-1 gene product. A short hydrophobic secretory sequence is first removed
to produce proendothelin-1. This is then cleaved at dibasic amino acid pairs by the
endopeptidase furin, generating the 39 amino acid peptide big endothelin-1 (Yanagisawa et
al., 1988). Subsequent production of mature endothelin-1 by a proteolytic cleavage between
Trp21 and Val22 is catalyzed by the membrane bound metalloprotease endothelin-converting
enzyme-1 (ECE-1). Although additional ECE isoforms have been identified in animals, a
human ECE-2 and ECE-3 have yet to be identified (Emoto et al., 1995). ECE gene knockout
studies suggest that ECE-1 is the major functional ECE for all three endothelin isoforms in
vivo (Yanagisawa et al., 1996). Endothelin-1 was initially considered to be produced de
novo in response to the factors described earlier. However, secretory vesicles containing
both mature endothelin-1 and ECE have been identified in endothelial cells (Turner et al.,
1996). Recently, a further endothelin peptide has been identified in humans formed by the
cleavage of big endothelin-1 at the Tyr31 and Gly32 bonds by a human chymase enzyme
expressed in mast cells. This product has been termed endothelin-11–31 though its role in
vivo has yet to be determined (Nakano et al., 1997).
ENDOTHELIN CONVERTING ENZYME
ECE-1 was first isolated and purified by Ohnaka and colleagues (1990) from aortic
endothelial cells. It is inhibited by the combined ECE and neutral endopeptidase (NEP)
inhibitor phosphoramidon, but not by selective NEP inhibitors such as thiorphan or
kelatorphan. Structurally, ECE-1 exists as a transmembrane 758 amino acid dimer, linked
by a single disulphide bridge. A short (1–56) N-terminal intracellular region is followed by
a 21 amino acid transmembrane region. A zinc-binding and catalytic site (595–599) is
essential for enzymatic activity. ECE-1 belongs to a family of neutral metalloprotease
enzymes which includes NEP and the human Kell blood group protein (Xu et al., 1994).
However, ECE is unique amongst this group in that it recognises a relatively long C-terminal
portion of big endothelin-1 (residues His27 to Gly34) in addition to the cleavage site between
residues 21 and 22 (Takayanagi et al., 1998).
The ECE-1 gene is located on chromosome 1 at the p36 band (Valdenaire et al., 1995).
cDNA cloning studies have demonstrated differential gene splicing leading to the production
of two isoforms of ECE-1, termed ECE-1a and ECE-1b, which differ in structure only at
the N-terminus. ECE-1a is responsible for generation of the majority of functional endothelin-
1 from big endothelin-1. ECE-1a is expressed by endothelial cells and is located
intracellularly, the enzymatically active C-terminal segment facing the intra-luminal region
of the Golgi apparatus. A generator role for ECE-1a is further suggested by the presence of
characteristic promoter regions for this gene, indicating that it is a constitutively expressed
‘housekeeping’ gene.
In contrast, ECE-1b spans the plasma membrane of effector cells, such as vascular smooth
muscle cells, converting extracellular big endothelin-1 to endothelin-1. A ‘responder/
regulator’ role for ECE-1b to extracellular big endothelin-1 is suggested by its promoter
region containing potential receptor sites for transcription factors, allowing modulation of
activity. Transfection of preproendothelin-1 and ECE-1b genes into cultured cells
demonstrates that ECE-lb expressed at the cell surface is relatively inefficient at proteolysis
of exogenous big endothelin-1, with only around 10% converted to endothelin-1. In contrast,
between 50–90% of the endothelin peptides secreted were in the mature endothelin-1 form
(Xu et al., 1994). This suggests that endogenously generated endothelin-1 secreted
abluminally is the most functionally important source and confirms a predominantly
autocrine/paracrine mechanism of action for endothelin-1. Such a theory is supported by
the low (<5pM) concentrations of endothelin-1 in the plasma, concentrations probably
insufficient to activate endothelin receptors. Concentrations of angiotensin II and atrial
narriuretic peptide in plasma are normally up to ten times greater than those of circulating
endothelin-1. Also, endothelin-1 has a half-life of less than five minutes in plasma, with
clearance occurring mainly in the lungs and kidneys (Dupuis et al., 1996). It is likely that
much higher concentrations of endothelin-1 occur at the junctions between endothelial and
vascular smooth muscle cells and that at least some of the plasma endothelin-1 represents
overspill from this site. One might conclude, therefore, that plasma levels of endothelin-1
in pathological states represent an unreliable index of vascular endothelin activity (Goddard
and Webb, 1999). Similarly, urinary concentrations of endothelin-1 may reflect local renal
endothelin activity better than they reflect systemic changes.
ENDOTHELIN RECEPTORS
The isoforms of endothelin exert their physiological effects in a receptor-mediated fashion.

Pharmacological analysis of this process suggested the existence of at least two endothelin
receptor types in humans, termed ETA and ETB receptors, and this has been confirmed by
molecular characterisation studies (Arai et al., 1990, Sakurai et al., 1990, Sakamoto et al.,
1991). ETA receptors are located on vascular smooth muscle cells (Arai et al., 1990, Hori et
al., 1992) and, when activated, produce a sustained vasoconstriction that is of slow onset.
In contrast, ETB receptors are located on both endothelial (Hosada et al., 1991, Molenaar et
al., 1993) and vascular smooth muscle cells (Davenport et al., 1993). Activation of ETB
receptors on endothelial cells causes vasodilatation (Takayanagi et al., 1991) through the
release of dilator mediators acting on smooth muscle cells, whilst activation of ETB receptors
on smooth muscle cells produces vasoconstriction directly (Williams et al., 1991, Moreland
et al., 1992, Sumner et al., 1992). There is some pharmacological evidence to suggest
subdivision within the ETA and ETB receptor types, derived from comparison of binding
affinities, agonist/antagonist potencies and cell signalling following agonist binding. At a
molecular level, however, there is currently no evidence to support this approach. The agonist
binding characteristics of the endothelin receptors are shown in Table 2.1.
Endothelin Receptor Genes

The human EDNRA gene is located on chromosome 4 and consists of eight exons and
seven introns spanning 40 kilobases (Hosada et al., 1992). The human EDNRB gene on
chromosome 13 is somewhat smaller in size, spanning 24 kilobases and consisting of seven
Table 2.1 Properties of the endothelin receptor subtypes.

exons and six introns (Arai et al., 1993). Analysis of cDNA clones predicts ETA and ETB
receptors consisting of 427 and 442 amino acids respectively and a sequence homology of
~58%. In a manner analogous to the preproendothelin-1 genes, both the ETA and ETB receptor
genes have promoter regions which control gene transcription levels in response to factors
such as nuclear factor-1, RNA polymerase II transcription factor and acute phase reactant
regulatory elements. The receptor genes also encode regions for the post-translational
modification of the receptors, altering tertiary structure, membrane anchorage sites and
linkage to intracellular effector mechanisms (Elshourbagy et al., 1993).
Distribution of Endothelin Receptor Genes

Autoradiography using labelled endothelin-1 and gene probes for the detection of endothelin
receptor mRNA has allowed the qualitative and quantitative assessment of both the tissue
distribution and the receptor subtype expressed in various tissues and cell lines. Such analysis
has demonstrated the localisation of ETA receptors predominantly to the vascular smooth
muscle cells of large and medium-sized arteries, the highest densities being found in the
aorta. In addition, renal arterioles, bronchial smooth muscle and glandular tissues, such as
those of the pituitary and adrenal glands, have been shown to preferentially express ETA
receptors (Arai et al., 1990). In contrast, the liver and endothelial cells are devoid of ETA
receptors (Hosada et al., 1991). ETB receptors are found on both smooth muscle and vascular
endothelial cells, particularly in the brain, lungs, liver, kidney, bowel and adrenal glands.
Human kidneys express ETB receptors as the dominant receptor subtype with the collecting
ducts exhibiting the highest density of ETB receptors (Hori et al., 1992). Human cardiac
tissue expresses both ETA and ETB receptors throughout the A-V node, His-bundle,
myocardium and endocardium (Molenaar et al., 1993). Differential receptor subtype
expression is thought to account for the wide variety of responses to the endothelins seen in
different cell types and tissues.
The pattern of receptor expression in different tissues may reflect local differences in
the transcription factors acting upon promoter regions to alter receptor mRNA expression.
Alternatively, up- or down-regulation of receptor numbers could be mediated by local
environmental factors such as endothelin-1 exposure or insulin concentrations, either causing
receptor internalisation or directly altering gene transcription. It has been proposed that
alterations in the spectrum or density of receptor expression by such mechanisms may be
responsible for certain pathological states. For example, Wang and colleagues (1996) have
used reverse transcription-polymerase chain reaction to quantitatively examine mRNA
expression in the rat carotid artery balloon angioplasty model. They demonstrated a two-
fold increase in preproendothelin-1 and ECE mRNA expression and a 30-fold increase in
both ETA and ETB receptor mRNA expression post-angioplasty. Neointimal lesions also
showed increased endothelin-1 immunoreactivity, suggesting a role for altered ETA and
ETB receptor expression, in addition to altered endothelin-1 expression, in the pathogenesis
of angioplasty-induced neointima formation. The precise role of changes in endothelin
receptor expression in humans, however, will require similar mRNA expression studies to
be performed on the relevant tissues.
Structure of Endothelin Receptors

The ETA and ETB receptors are part of the rhodopsin G-protein-coupled superfamily of
receptors. These share a structure consisting of an extracellular N-terminal region, seven

helical transmembrane loops connected by hydrophilic domains, and a 60 amino acid
intracellular carboxy terminal region. The hydrophobic transmembrane domains and the
interconnecting cytoplasmic loops are highly conserved, in contrast to the N-terminal region,
which shows only 4% sequence homology between the ETA and ETB receptors (Elshourbagy
et al., 1993). Investigation into the structural determinants of receptor function has been
achieved using the techniques of site directed mutagenesis of specific amino acid residues
and the formation of receptor chimeras.
N-terminus
The amino acid residues which lie nearest the first transmembrane region seem to be
essential for endothelin-1 binding. In particular, the Asp75 and Pro93 in this region of
the ETB receptor are thought to be responsible for the high stability of the complex
formed between endothelin-1 and the ETB receptor, compared to the ETA receptor
(Takasuka et al., 1994).
Transmembrane domains
The transmembrane regions I, II, III, and VII are the major determinants of ligand binding.
Ligand selectivity appears localised to transmembrane regions IV, V and VI and their
intervening loops. The boundary region between the first extracellular loop and the second
transmembrane domain regulates BQ-123 binding, whist the Lys140 in this area seems to be
necessary for the binding of endothelin-1 to the ETA receptor, possibly via the mediation of
conformational changes (Adachi et al., 1994).
Cytoplasmic loops
The cytoplasmic loops are known to mediate receptor/G-protein coupling in other
rhodopsin-like G protein receptors. Substitution of the C-terminal end of the third
cytoplasmic domain with the corresponding section of the (ß2-adrenoceptor results in no
changes in the affinity of the ETA receptor for endothelin-1, but receptor/ligand binding
fails to elicit the subsequent rise in intracellular calcium ([Ca2+]i) that normally follows
receptor activation (Adachi et al., 1993).
C-terminus
The C-terminal amino acids are thought to be responsible for anchorage of the endothelin
receptor to the plasma membrane. They may also mediate elements of signal transduction,
as evidenced by the loss of [Ca2+]i increases on ligand binding following removal of C-
terminal amino acids (Adachi et al., 1993).
Signal Transduction
Binding of endothelin-1 to the ETA receptor on vascular smooth muscle cells initiates a
complex cascade of events resulting in a biphasic rise in [Ca2+]i, and, ultimately, cellular
contraction and mitogenesis. Typically, the contractions induced by endothelin-1 develop
Figure 2.3 Signal transduction pathways of the endothelin receptors. MAPK=mitogen activated protein kinase.
Adapted from Decker & Brock, 1998 with kind permission of the authors. See text for details.
slowly but are sustained and resistant to agonist removal. These effects are mediated by
the interaction of the endothelin receptor with specific guanine nucleotide regulatory
proteins (G-proteins) in the cell membrane (Bitar et al., 1992). G-proteins are
heterotrimeric structures involved in receptor signal transduction for a wide variety of
cellular processes. Evidence from G-protein inhibitors such as Bordatella pertussis toxin
and Vibrio cholera toxin suggest that the endothelin receptor is able to link with a variety
of different G-protein subtypes. These include both inhibitory and stimulatory G-proteins
which are able to activate an array of inrracellular effector mechanisms, resulting in a
wide variety of biological responses. These second messenger systems are summarized
below and illustrated in Figure 2.3. The precise signalling mechanisms utilised by the
ETA and ETB receptors may exhibit subtle differences, although a consensus opinion in
this area is awaited (Douglas et al., 1997).
Phospholipase C
G-protein stimulated activation of phospholipase C (PLC) causes hydrolysis of
phosphatidylinositol 4,5-bisphosphonate (PIP2) to produce inositol 1,4,5-triphosphate (IP3)
and sn1,2-diacylglycerol (DAG). The rise in IP3 concentration stimulates release of
intracellular Ca2+ stores from the sarcoplasmic reticulum via both ryanodine- and IP3-sensitive
Ca2+ channels and is responsible for the rapid initial rise in [Ca2+]i seen following endothelin-
1 binding (Marsden et al., 1989). DAG production initiates activation of protein kinase C
(PKC) allowing sensitisation of the cellular contractile elements to changes in [Ca2+]i (see
below). The precise G-protein connecting the endothelin-receptor to phospholipase C is
currently unknown.
Calcium
As already indicated, endothelin receptor activation results in a rapid initial rise in
[Ca2+]i followed by a plateau phase dependent on the presence of extracellular Ca2+.
However, although usual, a rise in cellular IP3 via PLC activation is not mandatory to
cause a rise in [Ca2+]i. Endothelin-3 and SX6c acting on ETB receptors appear to cause
an IP3-independent rise in [Ca2+]i in certain cell types in which both the initial and
plateau phases of Ca2+ mobilisation are dependent on movement of Ca2+ into the cell
(Little et al., 1992).
Extracellular Ca2+ entry appears to be necessary for the sustained increase in [Ca2+]i
that permits prolonged cellular contraction in response to endothelin-1. Both voltage
operated and receptor operated calcium channels are involved in this process. L-type
calcium channel blockers (dihydropyridine class) attenuate the sustained rise in [Ca2+]i
by antagonism of voltage operated calcium channels. Prolonged cellular contraction may
also be prevented by nickel ions, which selectively block receptor operated channels.
The T-type calcium channel is a putative candidate for the receptor operated channel
(Stasch et al., 1989). The opening of voltage dependent L-type calcium channels is
dependent upon prolonged membrane depolarisation. The precise mechanism for achieving
and maintaining such a period of membrane depolarisation remains controversial but is
likely to involve calcium-activated chloride channels with resultant efflux of chloride
ions (James et al., 1994), and/or an influx of cations through non-selective cation channels.
Other candidates for the mediators of the increased [Na+]i that produces membrane
depolarisation include ATP-sensitive K+ channels, the Na+/K+-ATPase, or activation of
the Na+/H+ antiporter (Meyer-Lehnart et al., 1989, Danthaluri and Brock, 1990, Miyoshi
et al., 1992).
Protein kinase C
PKC exists in multiple isoforms and is one of the key regulatory enzymes involved in the
endothelin-1 signalling process. Activation of the endothelin receptor stimulates a rise in
DAG levels which, along with phosphatidylserine and Ca2+, results in the activation and
translocation of PKC from the cytosol to membranes within vascular smooth muscle cells.
Activated PKC then catalyses the phosphorylation of heavy and light myosin chains along
with caldesmons, which thereby increases the sensitivity of the contractile elements to [Ca2+]i
(Nishimura et al., 1992). Specific PKC inhibitors block the sustained contractile responses
to endothelin-1 and reduce Ca2+ sensitivity. PKC also modulates the hydrolysis of PIP2
following activation of endothelin receptors by exerting an inhibitory effect on PLC and
may form part of a negative feedback loop controlling PLD and arachidonic acid metabolism.
The mitogenic effects of endothelin-1 appear to be related to both PKC- and tyrosine kinase-
dependent mechanisms
Phospholipase D
The rise in intracellular DAG levels in response to endothelin-1 occurs in a biphasic
manner, the initial rise resulting from PIP2 hydrolysis by PLC. The prolonged secondary
rise in DAG levels is thought to originate from phosphatidylcholine hydrolysis mediated
by PLD and lasts for up to 20 minutes. Activation of PLD occurs via PKC-dependent
and PKC-independent mechanisms (Liu et al., 1992). The phosphatidic acid (PA)
produced enhances Ca2+ influx and further enhances PLC activity (Shukla et al., 1991).
It is proposed that PLD activation may contribute to the mitogenic effects of endothelin-
1 (Boarder, 1994).
Phospholipase A2
Phospholipase A2 (PLA2) catalyses the generation of arachidonic acid metabolites
including the leukotrienes, thromboxane A 2 (TxA2) and prostacyclin (PGI 2) from
membrane lipids. Inhibitors of the arachidonic acid metabolism cascade have been shown
to inhibit many of the actions of endothelin-1, and PGI2 production by endothelial cells
can be stimulated by endothelin-1. Linkage of endothelin-receptors to PLA2 may be direct
via a G-protein, or occur in response to endothelin-1-activated changes in [Ca2+]i (Barnett
et al., 1994).
Tyrosine kinases
Endothelin-1-induced tyrosine phosphorylation of cellular proteins modulates activation
of PLD and in part regulates its mitogenic effects. Stimulation of ETA receptors results in
phosphorylation of tyrosine residues on cytosolic proteins ranging in size from 45–225
kDa by members of the tyrosine kinase family known as Src, focal adhesion kinase (FAK)
and Janus kinase (Jak). Although the precise mechanisms are as yet unknown, it seems
likely that activation of transcription factors by these kinases in response to endothelin-1
stimulation is involved in the mitogenic response.
Nuclear/mitogenic signalling mechanisms

Alone, endothelin-1 is a weak mitogen, as measured by thymidine incorporation or
expression of the proto-oncogenes c-jun and c-fos. However, when combined with other
growth factors such as platelet derived growth factor (PDGF) and epidermal growth factor
(EGF) or co-mitogens such as insulin, it acts synergistically to produce marked mitogenic
effects. As indicated earlier, a precise mechanistic model is still awaited, but both phorbol
ester-sensitive PKCs and tyrosine kinase signal transduction mechanisms are involved.
PKCs, activated by the binding of endothelin to its receptor, catalyse the c-Raf-1 cascade
leading to activation of mitogen activated protein kinases (MAPKs). MAPKs are known
to be critically important in the transduction of mitogenic signals in a number of other

growth factor systems. Where the mitogenic response pathways of endothelin-1 differ
from those of other growth factors, however, is in the proposed coupling of the activated
ETA receptor to the c-Raf-1 cascade via PKC. Endothelin-1 activation of the tyrosine
kinases Src, FAK and JAK occurs in a non-PKC dependent manner. Their activation may
result in the switching on of transcription factors of the signal transducers and activators
of transcription family (STAT) and subsequent translocation of STATs to the nucleus
where they interact with promoter regions to initiate gene transcription (reviewed by
Decker and Brock, 1998). The study of the mitogenic responses to the endothelins is
currently in its infancy when compared to that of other aspects of endothelin physiology.
However, the similarities in the effector mechanisms utilised in response to endothelin-1
and other growth factors provide interesting parallels. ETA and ETB receptor activation is
also known to increase the expression of cellular adhesion molecules, induce chemotactic
factors such as TNFα, IL-1ß, IL-6, and IL-8, and alter matrix synthesis (reviewed by
Douglas and Ohlstein, 1997). Thus, in addition to short-term actions upon cellular
contractility, the endothelin isoforms also influence cellular growth and differentiation, a
process critically linked with the proposed role of endothelin-1 as a mediator of vascular
hypertrophy and cardiac remodelling seen in conditions such as hypertension and chronic
cardiac failure.
Nitric oxide and cGMP

The ETB receptor present on endothelial cells is responsible for vasodilatation in response
to activation by endothelin-1, endothelin-3 and the selective ETB receptor agonist SX6c.
This effect is produced by ETB receptor-stimulated release of the vasodilating factors
nitric oxide (NO) and PGI2. Activation of ETB receptor-coupled G-proteins leads to an
increase in PLC activity with generation of IP3 and DAG. This releases Ca 2+ from
intracellular stores (see above) with a resultant increase in the activity of a constitutive
Ca2+/calmodulin dependent NO synthase enzyme. Liberated NO diffuses to the smooth
muscle cell layer and initiates production of cGMP by soluble guanylate cyclase. Increasing
levels of cGMP activate mechanisms that ultimately decrease [Ca2+]i leading to cellular
relaxation (Lincoln, 1989). Inhibition of NO synthase by L-NG-monomethyl arginine (L-
NMMA) has been shown to inhibit the vasodilation in response to ETB receptor stimulation.
Further mechanisms of NO production have been proposed. These include a PKC-
dependent pathway and direct production of cGMP via activation of a pertussis toxin-
insensitive G-protein by endothelin-1 on the kidney epithelial cell line LLC-PK1 (Ozaki
et al., 1994). NO also exerts an inhibitory effect on endothelin-1 stimulated receptor
signalling. In Chinese hamster ovary (CHO) cells transfected with ETA receptors it has
been shown that NO can displace endothelin-1 from ETA receptors and disrupt calcium
mobilisation (Aramori et al., 1992).
Adenylate cyclase
Endothelin-1 may directly inhibit the accumulation of cAMP induced by forskolin, cholera
toxin and isoproterenol in endothelial cells (Ladoux et al., 1991). It is at present unclear
whether the effects of endothelin-1 on cyclic nucleotide levels are mediated directly via
adenylate cyclase or indirectly as a result of PLC activation.
Formation and Internalisation of Receptor Complexes

Early studies of endothelin-1-induced responses demonstrate a slow onset of action
followed by a period of sustained vasoconstriction that resists agonist removal by
prolonged washout. This characteristic pattern of vasoconstriction to endothelin-1 is a
product of both the ligand/receptor binding relationship and the signal transduction
mechanisms thereby induced. Endothelin-1 is a potent vasoconstrictor agent. Saturation
of endothelin receptors occurs within one minute of exposure to endothelin-1 at 37°C
(Marsault et al., 1991). Once bound, the binding of endothelin to its receptor appears to
be a ‘pseudo-irreversible’ process, with a dissociation half-life in excess of 100 hours
(reviewed by Douglas and Ohlstein, 1997). Rapid receptor complex internalisation by a
clathrin-mediated endocytic pathway follows, and, once internalised, degradation of the
endothelin/endothelin-receptor complex is thought to occur within lysosomes where the
local acidic environment favours dissociation. Re-cycling of receptors back to the cell
surface then occurs, allowing the cell to regulate the degree of endothelin-1 stimulation.
Degradation of receptor complexes by neutral endopeptidases located within the plasma
membrane may also occur prior to internalisation (Loffler et al., 1991). Prior to
internalisation, however, rates of dissociation of receptor and ligand are dependent upon
the duration of exposure, with studies using radio-labelled endothelin-1 showing a
progressive decline in the proportion of dissociable endothelin-1 over 60 minutes (Wilkes
and Boarder, 1991). Dissociable endothelin-1 is thought to represent the presence of
surface bound/non-internalised receptor complexes. There does appear to be some
heterogeneity amongst the different receptors and endothelin isoforms in the rate and
extent of receptor internalisation. For example, in anterior pituitary cells, the rate of
receptor endocytosis is higher for endothelin-1 bound to ETA receptors than for endothelin-
3 bound to the same receptor (Stojilkovic et al., 1992). Overall, the delayed action of
endothelin-1 appears to result from the time required to activate the receptor-coupled
second messenger systems outlined earlier, as opposed to delays in ligand/ receptor
binding, whilst the prolonged duration of action may be due to continued receptor
signalling following internalisation.
Receptor Downregulation
Repeated exposure of smooth muscle cells to endothelin results in the downregulation
of receptors without a change in their affinity. This is manifest as a progressive decrease
in tissue or cellular responsiveness (Hirata et al., 1988). This phenomenon occurs in
response to both exogenous and endogenously produced endothelins and receptor
downregulation is agonist selective. Pre-treatment of cells with the ETB receptor-selective
agonist SX6c results in the loss of further ETB receptor-agonist-mediated responses,
but the response to ETA receptor agonists is preserved (Henry, 1993). Interestingly,
stimulation with endothelin-3 of cells having no ETB receptor binding sites results in a
loss of further responsiveness to endothelin-3 but maintenance of endothelin-1-
stimulated responses (Hiley et al., 1992). This suggests that endothelin-3 may selectively
desensitise the ETA receptor to endothelin-3 but not endothelin-1. Alternatively, it has
been suggested that endothelin-3 may mediate its effects via a non-ETA and non-ETB
receptor (Hiley et al., 1992), although no molecular evidence for such a receptor has
yet emerged.
Endothelin Receptor Subtypes

Two further subtypes of endothelin receptor have been isolated in Xenopus laevis but not
from mammalian species. The first of these receptor subtypes, termed ETAX, displays
agonist binding characteristics identical to the ETA receptor but is insensitive to the selective
ETA receptor antagonist BQ-123 (Kumar et al., 1994). The second, isolated only from
Xenopus laevis melanocytes, has a greater binding affinity for endothelin-3 than
endothelin-1 and may represent a third morphological receptor subtype called ETC (Karne
et al., 1993). The Xenopus ETC receptor shares a 50% amino acid sequence homology
with the human ETA and ETB receptors and complementary DNAs for each of these
receptor subtypes have now been isolated and characterised. Analysis of human genomic
DNA with cDNA probes of low stringency has, as yet, failed to identify any further
homologous sequences. Any putative endothelin-3-selective ETC receptor would, therefore,
be likely to have a structure that differs widely from the other endothelin receptors
(Sakamoto et al., 1991).
Sub-division of the ETB receptor type has also been proposed. This stems from the
observation that activation of ETB receptors may produce either vasodilatation or
vasoconstriction. Vasodilatation in response to ETB receptor stimulation is mediated by
ETB receptors located on vascular endothelial cells through the production of nitric oxide
or dilator prostanoids (Takayanagi et al., 1991). In contrast, vasoconstrictor responses are
mediated directly by ETB receptors located on vascular smooth muscle cells (Williams et
al., 1991, Moreland et al., 1992, Sumner et al., 1992). However, despite reports that
production of two receptor subtypes from a single gene is possible by varying transcription
initiation sites or post-translational modification (Shyamala et al., 1994), it is more likely
that these differing responses are due to differences in signal transduction pathways in the
effector cells.
Pharmacological differences in the responses of ETA receptors in various tissues to
antagonists such as BQ-123 (a selective ET A receptor antagonist) have also been
demonstrated (Sudjarwo et al., 1994). This has led to suggestions that subtypes of the ETA
receptor may also exist though, again, validation by molecular studies has not yet been
forthcoming.
DEVELOPMENTAL BIOLOGY
Targetted gene knockout studies have provided further clues to the role of the endothelin
isoforms. Studies originally conceived to examine the physiological effects of deletion of
the genes for endothelin-1, ECE and the ETA and ETB receptors in the adult animal have
produced unexpected results. Mouse embryonic stem cells manipulated to carry a mutant
endothelin-1–allele underwent homologous recombination to produce endothelin-1-/- mice.
The offspring were characterised by lethal abnormalities of the craniofacial and pharyngeal
pouch structures. These structures are derived from the neural crest ectomesenchymal cells
indicating that endothelin-1 is crucially involved in normal ontogeny of the pharyngeal
arches (Kurihara et al., 1994). Targetted disruption of the ECE-1 gene produces identical
phenotypic abnormalities of the craniofacial structures, as does deletion of the ETA receptor
gene. Deletion of the ETB receptor or endothelin-3 gene produces mice characterised by
aganglionic megacolon and coat colour spotting (Hosada et al., 1994). The ET B
receptor/ endothelin-3 interaction therefore appears to be critical for the normal development
of epidermal melanocytes and enteric neurones. Additionally, ECE-1 knockout mice include
the phenotype of endothelin-3 knockout mice, suggesting that ECE-1 is functionally
responsible for the conversion of big endothelin-3 to endothelin-3 (Baynash et al., 1994).
Two human conditions share phenotypical similarities to endothelin-knockout mice models.
The first, the Pierre-Robin and Treacher-Collins syndromes share the same craniofacial
abnormalities as endothelin-1 or ETA receptor-knockout mice, indicating that the human
condition might occur secondary to ETA receptor/endothelin-1 anomalies (Ong, 1996).
Secondly, abnormalities of preproendothelin-3 or ETB receptor genes have been documented
to occur in the neurocristopathies associated clinically with Hirschsprung’s disease
(Puffenberger et al., 1994) and the Waardenburg-Shah syndrome (Attie et al., 1995)—both
of which are varieties of aganglionic megacolon. This has obvious implications for the
clinical use of ETA receptor antagonists and ECE-inhibitors, rendering them unsuitable for
use during pregnancy or at the time of conception, these abnormalities having been detected
in teratogenicity studies with endothelin antagonists in animals.
It is interesting to note that endothelin-1-knockout mice are hypertensive—at odds with
the predicted role of endothelin-1 as a mediator of vasoconstriction and contributing to
basal vascular tone. The explanation for this is thought to lie in the sympathetic-adrenergic
overactivity that is induced by hypoxia secondary to the severe craniofacial abnormalities,
or due to disruption of the central control of cardiorespiratory function, in which endothelin-
1 is thought to play a role (Kurihara et al., 1994).
EFFECTS OF ENDOTHELIN ADMINISTRATION
The endothelins act in a predominantly autocrine and paracrine fashion. Studies involving
the administration of the endothelin isoforms, either by bolus or constant infusion are,
therefore, unlikely to reproduce the in vivo physiological effects of endothelin action. Indeed,
such studies may be frankly misleading, despite the presence of increased circulating
concentrations of endothelin-1 having been documented in a number of pathological
conditions. For example, increased plasma concentrations of endothelin-1 in any given
disease state may result in downregulation of endothelin receptors with a resultant reduction
in the magnitude of responses to exogenous endothelin-1, in contrast to the predicted outcome
of higher endothelin-1 concentrations producing an amplification of the normal physiological
response. Here, studies with antagonists are more revealing. However, agonist studies are
helpful in defining target organs and the receptor subtype involved in the response, and
these data will, therefore, be reviewed below.
Effects of Endothelin on Blood Pressure and the Heart

Administration of endothelin-1 in humans by constant infusion produces a dose-dependent
increase in blood pressure that is of prolonged duration and accompanied by sodium retention
(Vierhapper et al., 1990). The sustained presser effect is also seen with bolus endothelin-1
administration, despite rapid clearance of the peptide from the blood. Pretreatment with either
cyclosporine, nifedipine, or the cyclo-oxygenase inhibitor indomethacin has no effect on the
blood pressure response to endothelin-1 (Vierhapper et al., 1992). In cardiac studies, infusion
of endothelin-1 at 8 pmol/Kg/min to healthy human subjects produced a reduction in coronary
blood flow of ~30%, with coronary vascular resistance increasing by ~100% (Pernow et al.,
1996). Mean arterial pressure increased during infusion of endothelin-1 in this study by ~10
mmHg. Big endothelin-1 was shown to be as potent as endothelin-1 in producing these effects,
probably reflecting local cardiac, or systemic, conversion of big endothelin-1 to endothelin-1
rather than a direct action of big endothelin-1 itself. Endothelin-1 exerts a negatively inotropic
effect in vivo, impairs diastolic filling of both the right and left ventricles, decreases cardiac
output and reduces heart rate (Kiely et al., 1997), probably through a baroreceptor-mediated
reflex. The impairment of diastolic function was also found at doses insufficient to alter systemic
or pulmonary blood pressure. Sarafotoxin-containing bites from Actractaspis have been shown
to cause myocardial infarction (Tony and Bhat, 1995), and coronary vasospasm has been
demonstrated in vitro in response to SX6c (Bax et al., 1994).
Effects of Endothelin on the Renal Circulation

The human renal circulation is extremely sensitive to the vasoconstrictor effects of
endothelin-1 and has a high capacity for the conversion of circulating big endothelin-1 to
endothelin-1 (Ahlborg et al., 1994). Endothelin-1 administered to healthy human subjects
at 4 pmol/ Kg/min produces an ~25% decrease in renal plasma flow (RPF) and an ~45%
increase in renal vascular resistance, the effects lasting up to three hours (Weitzberg et al.,
1991). Prolonged low dose infusions of endothelin-1 (0.4 pmol/Kg/min for six hours)
generate ~45% decrease in RPF and an ~80% increase in filtration fraction, indicating a
more pronounced vasoconstriction of the efferent arteriole in vivo (Jilma et al., 1997). Renal
vasoconstriction and sodium retention may be reversed by nifedipine, although the increase
in filtration fraction remains unaltered (Kaasjager et al., 1995). Nifedipine has a mainly
preglomerular site of action and does not, therefore, reverse the significant vasoconstrictor
effect of endothelin-1 on the efferent arteriole. In vitro studies have also demonstrated
vasoconstriction of the arcuate and inter-lobar arteries in response to endothelin-1 (Edwards
et al., 1990). Although there are marked interspecies variations in the receptor subtype
mediating these effects, the localisation of the ETA receptor subtype to the vasculature in
the human kidney makes this the most favoured candidate (Karet et al., 1993). Indeed, in
clinical studies, endothelin-3 infusion (selective for the ETB receptor) failed to alter renal
haemodynamics and electrolyte excretion (Kaasjager et al., 1997).
In vivo, endothelin-1 administration consistently produces sodium retention in humans,
even at doses insufficient to affect RPF or glomerular filtration rate (GFR) (Rabelink et al.,
1994). In the study by Kiely and colleagues cited earlier, low, medium and high dose
endothelin-1 infusions produced a decrease in plasma renin activity, but no significant effect
on aldosterone levels.
Effects of Endothelin on the Pulmonary Circulation

Wagner and colleagues (1992) have examined the effects of relatively low dose endothelin-
1 infusion (4 pmol/Kg/min) on the pulmonary circulation. No changes in pulmonary
haemodynamics were observed despite endothelin-1-induced increases in systolic blood
pressure, total and splanchnic vascular resistance and decreases in hepatic blood flow. An
absence of effect on the pulmonary vascular tree following intra-pulmonary endothelin-1
infusion was also found in patients with borderline pulmonary hypertension and chronic
hypoxaemia, although the difference between arterial and venous oxygen concentrations
was markedly increased during endothelin-1 infusion (reviewed by Holm, 1997). In contrast,
increases in total pulmonary vascular resistance and mean pulmonary artery pressure have
been noted with endothelin-1 infusion in other studies, measured using Doppler
echocardiographic techniques (Kiely et al., 1997). The pulmonary circulation does appear
responsible for the short half-life of endothelin-1, removing 50% of infused endothelin-1.
Concentrations of circulating endothelin-1 are known to be increased in pulmonary
hypertension and correlate with the severity of the disease. This has been shown to be at
least partially due to reduced pulmonary clearance of endothelin-1 (Dupuis et al., 1998)
but may also reflect increased local production (Cacoub et al., 1997).
Effects of Endothelin on Forearm Blood Flow

Many studies have examined the effects of giving endothelin isopeptides to animals.
However, because of the marked and sustained vasoconstriction produced by endothelin-1,
particularly in the renal and coronary vascular bed, there have been fewer studies in humans.
The potential risks associated with administering systemic doses of endothelins to humans
have prompted a number of investigators to utilise the technique of forearm plethysmography
coupled with brachial artery administration of locally active doses of drug. This technique
allows precise assessment of drug effects on vascular smooth muscle in vivo, without the
confounding influences of drug effects on other organs or activation of neurohumoral
reflexes. Changes in blood flow may, therefore, be attributed solely to the drug infused.
Blood flow in the opposite arm can also be measured to provide a contemporaneous control
during such studies and increase their power (Webb, 1995).
Endothelin-1 infusion into the brachial artery in healthy humans produces a slow onset
of vasoconstriction that is dose-dependent. The effect lasts for up to two hours after
discontinuation of the infusion (Clarke et al, 1989). The infusion of ETB receptor agonists
such as endothelin-3 or SX6c also produces a reduction in forearm blood flow, albeit to a
lesser extent, suggesting that ETB receptors are able to mediate at least part of the
vasoconstrictor effect in human resistance vessels. A similar response is seen in forearm
capacitance vessels (Haynes et al., 1995). However, the relevance of this vasoconstrictor
response to the physiological role of ETB receptor-mediated vascular responses is uncertain.
The forearm vasoconstriction to endothelin-1 may be overcome by co-infusion of Ca2+
antagonists but is not modulated by NO (Kiowski et al., 1991).
Bolus administration of either endothelin-1, endothelin-3 or SX6c produces transient
vasodilatation before causing sustained vasoconstriction. Vasodilatation is more pronounced
and sustained with endothelin-3 and SX6c, indicating that it is likely to be mediated via the
endothelial ETB receptor (Inoue et al., 1989a, Ohlstein et al., 1990). A similar response is
found in humans (Haynes et al., 1995b). However, this is seen only following high doses
on bolus administration and is, therefore, likely to be a pharmacological rather than a
physiological phenomenon.
Effects of Endothelins on Hand Veins

Human dorsal hand veins are an attractive experimental model because they possess no
intrinsic tone and their lack of response to big endothelin-1 (Haynes et al., 1995a) suggests
that they do not constitutively express ECE. Stimulation with endothelin-1 produces
venoconstriction that is modulated by endogenous endothelial PGI2 activity but not nitric
oxide synthesis (Haynes and Webb, 1993a). Sarafotoxin S6c is also able to produce dorsal
hand vein constriction, indicating that ETB receptors may contribute to venoconstriction.
The venoconstrictor response is dependent on the endothelin-1-stimulated closure of K+ATP
channels causing membrane depolarisation, an effect reversed by the K+ATP channel opener
cromakalim (Haynes and Webb, 1993b). Dihydropyridine Ca2+ channels are also responsible
for signal transduction in hand veins but to a lesser extent.
Effects of Endothelin on the Skin Microcirculation

Injection of endothelin-1, but not endothelin-3, into the skin produces vasoconstriction of
the microcirculation (Wenzel et al., 1994). Co-injection of ETA receptor antagonists reverses
the effects of endothelin-1 and elicit vasodilatation when given alone, indicating that
endothelin-1 may contribute to basal tone in the microcirculation of the skin. A flare reaction
is produced surrounding the central area of vasoconstriction at the site of injection of
endothelin-1. This flare results from the endothelin-1-stimulated release of nitric oxide,
possibly from polimodal nociceptor fibres located in the dermis, causing local vasodilatation
around the injection site (Wenzel et al., 1998). The authors suggest that endothelin-1 may,
therefore, be involved in the pathogenesis of neurogenic inflamation.
Effects of Endothelin on the Adrenals

Systemic infusion of endothelin-1 has no effect on basal aldosterone concentrations in healthy
men but does cause a rise in serum K+ concentration. However, there is selective augmentation
of aldosterone secretion in response to exogenous ACTH if endothelin-1 is administered
concomitantly (Vierhapper et al., 1995). In contrast, there is no augmentation of the ACTH-
induced secretion of cortisol, corticosterone or 18-OH-corticosterone, suggesting that
endothelin-1 exerts its effects in the later stages of steroid production.
Effects of Endothelin on the Pituitary

The pituitary gland contains an abundance of ETA receptors (Kanyicska and Freeman, 1993)
and the release of endothelin-3 from rat pituitary cells has been demonstrated (Matsumoto
et al., 1989). In humans, endothelin-1 infusion augments the corticotrophin releasing
hormone-induced rise in plasma ACTH levels and possibly also the responses to luteinising
hormone and follicle stimulating hormone induced by their respective releasing hormones.
In contrast, endothelin-1 inhibits the rise in prolactin, growth hormone and thyroid
stimulating hormone seen following stimulation by their releasing hormones. Basal hormone
levels are unaffected by endothelin-1 administration (Vierhapper et al., 1993). The plasma
concentrations of endothelin-1 achieved during these experiments were greater than
physiological but similar to those documented in pathophysiological states. The effects of
endothelin-1 upon the posterior pituitary are complex. In the rat, the paraventricular and
supraoptic nuclei of the hypothalamus and the axons of those neurons ending in the posterior
pituitary exhibit endothelin-containing secretory vesicles (Yoshizawa et al., 1990). These
vesicles become depleted during water deprivation. In man, upright tilt results in a parallel
rise in the concentrations of endothelin-1 and anti diuretic hormone (ADH). This response
is absent in patients with diabetes insipidus (Kaufmann et al., 1991). Further support for
the theory that endothelin-1 regulates the release of ADH comes from the observation that
infusion of endothelin-1 in dogs results in a rise in plasma ADH concentrations (Nakamoto

et al., 1989). Activation of the hypothalamic-pituitary axis by endothelin-1 may, therefore,
be clinically relevant.
CARDIOVASCULAR PHYSIOLOGY OF THE ENDOTHELINS
As already indicated, agonist studies are potentially poor predictors of the physiological
role of the endothelin peptides and the physiology of autocrine and paracrine systems is
best examined using antagonists. Studies with endothelin receptor antagonists and ECE
inhibitors have provided important insights into the role of the endothelin system in the
maintenance of basal vascular tone and also indicated important interactions with the
sympathetic nervous system and the L-arginine/nitric oxide system.
Local Cardiovascular Effects

One of the first available antagonists of the endothelin system was phosphoramidon, an
inhibitor of ECE-1 and neutral endopeptidase (NEP), and this was utilised to study the in
vivo role of ECE. Brachial artery administration of big endothelin-1 in healthy subjects
caused a dose-dependent forearm vasoconstriction that could be blocked completely by
phosphoramidon (30 nmol/min), suggesting that the effects of the precursor are mediated
through conversion to the mature peptide by ECE (Haynes and Webb, 1994). The blockade
of constriction to big endothelin-1 by phosphoramidon is unlikely to have been due to
inhibition of endothelin receptor binding as vasoconstriction to endothelin-1 was unaffected
by this dose of phosphoramidon and because conversion of infused big endothelin-1 to
endothelin-1 and its C-terminal fragment was confirmed in plasma samples taken from the
veins draining the infused forearm (Plumpton et al., 1995). Big endothelin-1 conversion in
the forearm presumably occurs via vascular, probably endothelial, ECE situated within the
forearm resistance vessels since circulating blood exhibits little ECE activity (Watanabe et
al., 1991). The difference in potency between big endothelin-1 and endothelin-1, and the
ratio of concentrations of C-terminal fragment to big endothelin-1 in venous blood, both
indicate that local ECE converts ~10% of luminally presented big endothelin-1 to endothelin-
1, consistent with ~10% conversion of exogenous big endothelin-1 by cells expressing the
ECE-1 gene (Xu et al., 1994). Big endothelin-1 does not cause venoconstriction in hand
veins (Haynes et al., 1995a), even though these vessels respond to endothelin-1 (Clarke et
al., 1989), suggesting that ECE activity may not be present in all vessel types.
Administration of phosphoramidon (30 nmol/min) alone results in a slowly progressive
vasodilatation, consistent with a role for endothelin-1 in maintenance of basal vascular
tone (Haynes and Webb, 1994) (Figure 2.4). Although phosphoramidon also inhibits NEP,
this latter action is unlikely to explain the vasodilatation because potent and selective
inhibitors of NEP, such as thiorphan and candoxatril, cause slowly progressive forearm
vasoconstriction (Ferro et al., 1998). This effect of NEP inhibitors is likely to be caused by
accumulation of endothelin-1, because it is a substrate for metabolism by NEP and the
vasoconstriction is blocked by local infusion of the ETA receptor antagonist BQ-123, but
not by systemic ACE inhibition. This observation may also account for the increase in
plasma endothelin by NEP inhibitors in clinical trials (Ando et al., 1995) and their failure
to lower blood pressure in hypertensive subjects (reviewed by Ferro et al., 1998).
Figure 2.4 Forearm vasoconstriction to brachial artery infusion of endothelin-1 (5 pmol/min; closed circles) is
abolished by the co-infusion of the ETA antagonist BQ-123 (100 nmol/min; open circles). Infusion of BQ-123
(100 nmol/min; open squares) or the ECE/NEP inhibitor phosphoramidon (30 nmol/min; closed ovals) alone
produce progressive forearm vasodilatation whereas the selective NEP inhibitor thiorphan (30 nmol/min; open
ovals) causes progressive vasoconstriction. Adapted from Haynes & Webb, 1994, with kind permission of the
Lancet. See text for details.
Using the forearm blood flow model, Haynes and Webb (1994) provided further evidence
supporting a role for endothelin-1 in the maintenance of basal vascular tone. Using the
selective cyclic pentapeptide BQ-123, they demonstrated inhibition of endothelin-1-induced
vasoconstriction with local ETA receptor antagonism and BQ-123 caused vasodilatation
when given alone. The vasodilatation was of slow onset but persisted for up to one hour
after the infusion was discontinued. These results have since been confirmed by others
(Berrazueta et al., 1997, Verhaar et al., 1998) and suggest that the contribution of endogenous
endothelin-1 to the maintenance of basal vascular tone is largely mediated via the ETA
receptor.
Systemic Cardiovascular Effects

The relevance of the observations with endothelin antagonists in the forearm model are
confirmed by the effects of systemically administered TAK-044, a combined ETA/ETB
receptor antagonist. TAK-044 causes a reduction of both blood pressure and peripheral
vascular resistance when infused into normotensive individuals (Haynes et al., 1996), with
a dose of 1000 mg over 15 minutes causing a 4% and 18% reduction of systolic and diastolic
blood pressures respectively, along with a 26% decrease in peripheral vascular resistance,
thus indicating that its main effect is on resistance vessels. The summation of the
physiological effects of the endothelin isoforms acting on both ETA and ETB receptors in
human resistance vessels in vivo is, therefore, one of vasoconstriction. Bosentan, a combined
ETA/ETB receptor antagonist, has also been shown to exert a modest hypotensive effect in
healthy subjects (Weber et al., 1996). Studies with bosentan given over four weeks to patients
with essential hypertension have also shown a hypotensive effect, comparable to that of
enalapril, indicating that the endothelin system contributes importantly to blood pressure
in hypertensive individuals (Krum et al., 1998).
Role of the ETB Receptor

Whether endothelial ETB receptor-mediated vasodilatation or vascular smooth muscle
ETB receptor-mediated vasoconstriction is the predominant in vivo response to basal
endogenous endothelin-1 is an important question relevant to the development of ETA
receptor-selective or combined ETA/ETB receptor antagonists. Indeed, the potentially
beneficial effects of NO release (with its vascular growth and platelet aggregation inhibiting
properties) and of vasodilatation produced by endogenous ETB receptor stimulation might
be lost with the use of truly non-selective antagonists or ECE-inhibitors. Verhaar and
colleagues (1998) have recently addressed this question in the human forearm. In
agreement with previous studies, they demonstrated an increase in forearm blood flow
following administration of the selective ETA receptor antagonist BQ-123. In addition,
this vasodilatatory response was shown to be largely mediated by, presumably increased,
generation of NO (see Figure 2.5). Inhibition of vascular ETB receptors with the selective
ETB antagonist BQ-788 produced vasoconstriction and, when co-infused with the ETA
receptor antagonist BQ-123, BQ-788 attenuated the vasodilator response (see Figure 2.6).
Thus, the summation of effects of endogenous activation of ETB receptors is one of
vasodilatation via endothelin-stimulated generation of NO. Dilator PGI2 generation
following ETB receptor activation appears to contribute little to this response because
pre-treatment with aspirin has no influence on the response. Interestingly, many
cardiovascular diseases have shown to cause dysfunction of the endothelium, with a
resultant decrease in the production of NO or an increase in its breakdown (reviewed by
Ferro and Webb, 1997). In such pathological states, the NO mediated vasodilator response
to endogenous ETB receptor activation may be lost, allowing unopposed vasoconstriction
to predominate. The preservation of ETB receptor mediated effects may be particularly
important in the renal circulation. As indicated earlier, the ETA receptor appears to be
responsible for the mediation of vasoconstrictor responses to endothelin-1 in the human
kidney. In contrast, animal studies suggest that ETB receptors located on the proximal
tubules are important in the control of diuresis and natriuresis (King et al., 1989) and
may mediate renal vasodilatation. Indeed, rescued ETB receptor knockout mice are
Figure 2.5 Slow onset of forearm vasodilatation in response to brachial artery infusion of BQ-123 (100 nmol/
min; closed circles) during coinfusion of saline. Inhibition of prostanoid generation during BQ-123 infusion (100
nmol/min) had no significant effect (open triangles). Vasodilator effects of BQ-123 (100nmol/min) were attenuated
by inhibition of NO (closed squares). Adapted from Verhaar et al., 1998, with kind permission of Circulation. See
text for details.
hypertensive secondary to renal retention of sodium and chronic treatment of rats with
selective receptor antagonists results in hypertension (reviewed by Webb et al., 1998).
Taken together, these findings suggest an important protective role for the ETB receptor
in the renal responses to endothelin-1.
Interactions Between the Endothelin System and the Nitric Oxide, Renin-
Angiotensin and Sympathetic Nervous Systems
It is well recognised that endothelin-1 release is inhibited by endothelium-derived nitric
oxide in the short term and that prolonged exposure to NO stimulates upregulation of
ETA receptors. Inhibition of the NO synthetic pathway by L-NMMA results in the
potentiation of the vasoconstrictor responses to endothelin-1 (Lerman et al., 1992) and
an increase in circulating endothelin-1 levels (Ahlborg et al., 1997). Recent studies in
pigs have confirmed a close interaction between the NO and endothelin systems. A high
cholesterol diet over a 10 week period resulted in enhanced coronary vasoconstriction in
Figure 2.6 Slow onset forearm vasodilatation in response to brachial artery infusion of BQ-123 (10 nmol/min;
closed circles) alone, was attenuated by co-infusion of BQ-788 (1 nmol/min; open circles). Infusion of BQ-788
alone caused a small but significant vasoconstriction (closed triangles). Adapted from Verhaar et al., 1998, with
kind permission of Circulation. See text for details.
response to pathophysiological doses of endothelin-1, whilst basal NO activity was

reduced, suggesting an overall disturbance of vascular reactivity in this experimental
model (Mathew et al., 1997). In addition, the important role of the endothelin ETB receptor
in modulating responses to endothelin-1 in resistance vessels in man has already been
described (Verhaar et al., 1998).
In vitro experiments with human blood vessels have shown a potentiation of
norepinephrine-stimulated vascular contractions when endothelin-1 is co-administered (Yang
et al., 1990) and sympathetically mediated venoconstriction of capacitance vessels may be
potentiated by endothelin-1 in hypertensive patients (Haynes et al., 1994). Norepinephrine
infusions in rats increase the expression of endothelin-1 mRNA in ventricular myocytes
and cause an increase in cardiac mass, an effect that may be blocked with the combined
ETA/ETB receptor antagonist, bosentan (Kaddoura et al., 1996).
Similarly, angiotensin II stimulates both increased expression of endothelin-1 mRNA
(Imai et al., 1992) and increased release of endothelin-1 from endothelial and vascular
Table 2.2 Cardiovascular and related conditions that have been associated with subtypes of endothelial dysfunction.
v
represents an association between these disease states and endothelial dysfunction affecting the particular pathway.
smooth muscle cells (Dohi et al., 1992, Sung et al., 1994). Angiotensin II-induced
vasoconstriction may be potentiated by endothelin-1 administration (Yoshida et al., 1992),
whilst the pressor and mitogenic effects of prolonged angiotensin II infusion are abolished
by concomitant infusion of ETA receptor antagonists (Moreau et al., 1997).
The close interactions between the endothelin, renin-angiotensin and sympathetic nervous
systems suggest important synergistic effects leading to the vascular hypertrophy and
increased peripheral vascular resistance seen in hypertension and chronic heart failure.
Endothelin antagonists may, therefore, be of use in the treatment of these conditions to
prevent the complications associated with overactivity of the renin-angiotensin and
sympathetic nervous systems and/or decreased activity of the NO system.
The vascular responses to NO and the sympathetic nervous system are characteristically
of rapid onset and are relatively short-lived. It may, therefore, be that NO and the sympathetic
nervous system provide minute-by-minute, whilst endogenous endothelin-1 secretion
controls long-term, maintenance of vascular tone. Endothelial dysfunction affecting the L-
arginine/NO system, allowing unopposed activity of the endothelin system, is a proposed
model for a range of pathological conditions. A number of these conditions are also associated
with altered sympathetic activity. Examples of conditions proposed to be associated with
endothelial dysfunction affecting the L-arginine/NO, endothelin and sympathetic nervous
systems, indicating their potential interactions, are given in Table 2.2.
PATHOPHYSIOLOGY OF ENDOTHELIN
The normal function of the endothelin system and the balance with other local and hormonal
cardiovascular regulatory mechanisms may become disrupted by a number of
pathophysiological mechanisms (see Table 2.3). Many cardiovascular diseases have been
Table 2.3 Proposed mechanisms of endothelial system dysfunction in

various pathological conditions.
shown to be associated with increased circulating concentrations of plasma endothelin-1,

though whether these represent primary or secondary phenomena is undetermined. Changes
in gene expression for the endothelin precursors, endothelin converting enzymes or
endothelin receptors may cause congenital conditions such as Hirschsprung’s disease and
the Waardenburg-Shah syndromes. More subtle genetic mutations may result in changes in
receptor expression, affinity or selectivity, perhaps contributing to hypertension in some
patients. Enhanced receptor number or affinity is thought to mediate the hypertension and
renal dysfunction during cyclosporine treatment, and renal dysfunction may reduce peptide
clearance, thereby increasing circulating endothelin concentrations. Enhanced production
of endothelin-1, in response to a number of factors including vasoactive hormones and
tissue hypoxia, may further augment the neurohormonal activation seen in chronic heart
failure and enhance the effects of other mediators. Indeed, the combined ETA/ETB receptor
antagonist bosentan has been shown to produce favourable haemodynamic responses in
the short term in patients with chronic heart failure in whom ACE inhibitors had been
withheld (Kiowski et al., 1995). In a similar context, the selective ETA receptor antagonist
BQ-123 elicits short term improvements in cardiac index, and produces decreases in both
systemic vascular resistance and mean arterial pressure, when administered to small groups
of patients with stable chronic heart failure also taking ACE inhibitors (Cowburn et al.,
1998).
The actions and concentrations of the endothelins would also be potentiated in conditions
characterised by dysfunction of the L-arginine/NO system, allowing uncompensated changes
in vascular tone and vascular remodelling to occur. This has been suggested as a mechanism
for Raynaud’s disease and cerebral vasospasm, amongst others. For a review of the
pathophysiology of endothelial dysfunction, readers are referred to the appropriate chapters
later in this book.
SUMMARY
Endothelin-1 is a 21-amino acid peptide produced by the vascular endothelium that has
potent and long-lasting vasoconstrictor effects. Responses to endothelin-1 are mediated
by two subtypes of endothelin receptor, ETA and ETB, acting in an autocrine and paracrine
fashion. Clinical studies demonstrate a central role for endothelin-1 in the regulation of
vascular tone in concert with the L-arginine/NO and sympathetic nervous systems. In
addition to its effects on vessel tone, endothelin-1 exerts mitogenic effects which may
contribute to the cardiac and vascular hypertrophy or remodelling central to the pathology
of hypertension and chronic cardiac failure. Endothelin-1 has also been implicated in a
wide variety of pathological conditions including renal, pulmonary, gastrointestinal and
neurological disorders (Ferro and Webb, 1997). The use of both peptide and non-peptide
receptor antagonists in experimental models of cardiovascular disease have provided
valuable insights into the potential therapeutic uses of these agents. Large scale clinical
trials are now underway in several different cardiovascular diseases, including
hypertension, heart failure and sub-arachnoid haemorrhage, to further assess their efficacy
and safety.
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Endothelial cells not only mediate relaxation, but may also produce vasoconstrictor substances in response to a
number of agents and physical stimuli. Of relevance is the activity of an endothelial cyclooxygenase pathway
which can produce thromboxane A2, prostaglandin H2 and oxygen free radicals, mainly superoxide anions. In
agreement with experimental evidence, cyclooxygenase-dependent endothelium-derived contracting factors were
at first identified as responsible for impaired endothelium-dependent vasodilation in patients with essential
hypertension. This alteration does not seem to be related to the increase in blood pressure, since it was not seen in
patients with hypertension secondary to primary aldosteronism or renovascular disease, who also have endothelial
dysfunction. In effect, production of cyclooxygenase-dependent endothelium-derived contracting factors is a
phenomenon which is specific to aging, with essential hypertension merely causing an acceleration and enhancement
of this alteration. It is worth noting that both in aging and hypertension appearance of cyclooxygenase-derived
contracting factors is associated with a parallel decrease in nitric oxide availability, suggesting that the contracting
factor could be an oxygen free radical. In line with this possibility, in essential hypertension both indomethacin, a
cyclooxygenase inhibitor, and vitamin C, an antioxidant, increase the vasodilation to acetylcholine by restoring
nitric oxide availability. Moreover the magnitude of effect of these two substances is similar and not additive,
consistent with the idea that cyclooxygenase might be a source of oxidative stress in human hypertension. Other
clinical conditions characterized by production of cyclooxygenase-dependent endothelium-derived contracting
factors are acute estrogen deprivation and heart failure. Thus in normotensive women, ovariectomy and acute
estrogen deprivation causes endothelial dysfunction resulting from production of cyclooxygenase-dependent
vasoconstrictor substances. Finally, in patients with heart failure, indomethacin increases forearm vasodilation to
acetylcholine, demonstrating that cyclooxygenase-dependent endothelium-derived contracting factors can play a
major role in determining endothelial dysfunction in this pathological condition.
Key words: cyclooxygenase, acetylcholine, nitric oxide, oxidative stress, aging, essential hypertension.
3 Cyclooxygenase-Dependent Endothelium-Derived
Contracting Factors
Stefano Taddei, Agostino Virdis, Lorenzo Ghiadoni and

Antonio Salvetto
Department of Internal Medicine, University of Pisa, Via Roma, 67, 56100 Pisa, Italy
Tel: +39-50-551110; Fax: +39-50-502617; E-mail: s.taddei@int.med.unipi.it
INTRODUCTION
Soon after the initial discovery by Furchgort and Zawadzki (1980) of the obligatory role of
endothelial cells in relaxations of rabbit isolated arteries to acetylcholine, De Mey and
Vanhoutte (1982; 1983) found that the endothelium can also induce contractions of isolated
canine arteries and veins. A large number of agents and physical stimuli produce such
contractions and, depending on the agent, stimulus, and anatomic origin of the blood vessel,
several different endothelium-derived contracting factors have been described, including
cyclooxygenase-dependent endothelium derived contracting factors (EDCFs), endothelin
and angiotensin II (Lüscher, 1990). Although relaxing factors play an important physiological
role in circulatory regulation, existing experimental evidence supports the concept that
contracting factors may become important regulators of vascular tone and structure in aging
or under pathological conditions such as hypertension, diabetes, vasospasm and reperfusion
injury (Katušic, 1991; Lüscher, 1991).
Among the different pathways leading to endothelium-dependent contractions, it became
apparent that cyclooxygenase could play a primary role. Arachidonic acid induces
endothelium-dependent contractions in arteries and veins which can be inhibited by
cyclooxygenase blockers (Miller, 1985; Katušic, 1998).
Moreover cyclooxygenase-dependent endothelium-derived contracting factors can be
also induced by acetylcholine and the calcium ionophore A23187 in different vessels in
experimental models of hypertension or diabetes (Konishi, 1983; Lüscher, 1986; Katušic,
1988; Tesfamarian, 1989).
So far in animal vessels, two kinds of mediators of cyclooxygenase-dependent EDCFs
have been identified including oxygen free radicals (mainly superoxide anions), generated
by the hydroperoxidase activity of the enzyme, and prostanoids such as thromboxane A2 or
prostaglandin H2 (Figure 3.1) (Lüscher, 1990). It is relevant to observe that while prostanoids
act exclusively as direct vasoconstrictors, oxygen free radicals can either directly constrict
vascular smooth muscle, possibly by acting on prostaglandin H2 receptors, or indirectly
since they enhance NO breakdown (Figure 3.1).
This brief review will focus on current knowledge of cyclooxygenase-dependent EDCFs
and their role in the control of vascular tone in humans.
63
64 S.Taddei et al.
Figure 3.1 Schematic diagram showing endothelium-derived nitric oxide (NO) and cyclooxygenase-dependent
contracting factors in the vessel wall. In certain conditions, including advancing age or essential hypertension,
endothelial stimulation can activate not only L-arginine-NO pathway, but also cyclooxygenase to produce and
secrete prostanoids such as thromboxane A2 (TX A2) or prostaglandin H2 (PG H2) and oxygen free radicals which
cause vasoconstriction. Oxygen free radicals are also potent NO breakdown inductors.
IDENTIFICATION OF CYCLOOXYGENASE-DEPENDENT EDCFS IN

HUMANS
Studies in humans have been conducted in the peripheral vasculature by using the technique
of forearm blood flow measurement. Intra-arterial infusion of agonists and antagonists at
concentrations which are inactive systemically allows adequate local vascular stimulation
to be obtained without systemic effects or stimulation of neurohormonal reflexes. In this
way, the increase or decrease in forearm blood flow, measured by strain gauge
plethysmography, is an index of local vasodilation and vasoconstriction.
The possible production of cyclooxygenase-dependent EDCFs in humans was first tested
in genetic and non-genetic models of secondary hypertension. Thus the response to
acetylcholine is impaired in the forearm circulation of patients with essential hypertension
as compared to normotensive controls (Linder, 1990; Panza, 1990; Taddei, 1993). In these
patients, but not in healthy controls, infusion of intrabrachial indomethacin, a cyclooxygenase
inhibitor, increases the response to acetylcholine (Taddei, 1993). This finding clearly indicates
the production of cyclooxygenase-dependent products which contribute to the pathogenesis
of endothelial dysfunction in human primary hypertension. These effects of indomethacin
are obtained with an infusion rate of 50 µg/100 ml forearm tissue/min, which should provide
a local plasma drug concentration of around 10-5 M. Although this is the concentration of
indomethacin often employed in animal experiments, it should be recognised that at these
doses the drug is no longer selective. However when we tested different indomethacin
Cyclooxygenase-Dependent Endothelium-Derived Contracting Factors 65
Figure 3.2 Acetylcholine-induced increase in forearm blood flow (FBF) in the presence of saline (0.2 ml/min) or
indomethacin at 5, 15, 50 and 100 µg/100 ml forearm tissue/mm in essential hypertensive patients (n=6 each).
Data are shown as means ±SD and expressed as absolute values. * denotes a significant difference between
infusion in control conditions and in the presence of different infusion rates of indomethacin (p<0.05 or less)
(adapted from Taddei, 1998).
infusion rates to titrate the lowest and highest effective dose, we observed that an
indomethacin infusion rate of 5 µg/100 forearm tissue/min, 10 times lower than the standard
dose, did not change the vascular response to acetylcholine (Figure 3.2) (Taddei, 1998).
Moreover the infusion rate of 50 µg/100 ml forearm tissue/min maximally potentiates the
vasodilation to acetylcholine (Figure 3.2). It is therefore crucial to employ this compound
at an adequate local concentration to be able to demonstrate the possible presence of
cyclooxygenase EDCFs, at least in essential hypertensive patients, since lower concentrations
such as those obtained with systemic administration would be devoid of any effectiveness
in blocking EDCF production.
Patients with hypertension secondary to primary aldosteronism or renovascular disease
are also characterized by suppressed endothelium-dependent vasodilation (Taddei, 1993).
However in these models of secondary hypertension, indomethacin did not improve the
response to acetylcholine, indicating that EDCFs play no role in determining endothelial
dysfunction in human secondary hypertension. Taken together these results are in agreement
with experimental findings, demonstrating that cyclooxygenase-dependent EDCFs are
produced in the presence of genetic hypertension, but not in the presence of secondary
hypertension. The major relevance of this information is that EDCF production is probably
not a consequence of the increase in blood pressure but is likely to be pathogenically related
to essential hypertension.
Despite its marked effect on vasodilation to acetylcholine in the forearm circulation
of essential hypertensive patients, intrabrachial infusion of indomethacin does not change
66 S.Taddei et al.
local basal flow. This finding indicates that cyclooxygenase-dependent EDCFs are not
tonically produced and therefore do not participate in the control of basal tone. Only
selective receptor-mediated endothelial activation can induce the production of these
substances.
ARE CYCLOOXYGENASE-DEPENDENT EDCFS CHARACTERISTIC OF

ESSENTIAL HYPERTENSION?
The finding that indomethacin does not improve the blunted vasodilation to acetylcholine in
patients with secondary hypertension seems to exclude the possibility that an increase in blood
pressure per se could be the main mechanism leading to production of such substances.
Moreover the possibility that EDCFs could be related to the pathogenesis of essential
hypertension seems to be excluded by the results obtained in young normotensive offspring
of essential hypertensive patients. These subjects showed an impaired response to acetylcholine
as compared to matched offspring of normotensive subjects, while vasodilation to sodium
nitroprusside was similar in the two groups (Taddei, 1996a). Thus a genetic predisposition to
develop hypertension is associated with impaired endothelium-dependent vasodilation,
suggesting that the alteration is a primary defect and is not dependent upon high blood pressure.
However, in contrast to older essential hypertensive patients, the alteration seen in the
normotensive offspring was not reversed by indomethacin infusion, but was sensitive to
administration of L-arginine, the substrate for NO-synthase (Taddei, 1996a). This might indicate
that a primary defect in the L-arginine-NO pathway, and not production of cyclooxygenase-
dependent EDCFs, is responsible for the endothelial dysfunction present in individuals with a
familial predisposition to develop hypertension. Thus EDCFs cannot universally participate
in endothelial dysfunction in essential hypertension.
Therefore, to identify the significance of cyclooxygenase-dependent EDCF production
in human cardiovascular physiopathology it is crucial to consider the impact of increasing
age on endothelium-dependent vasodilation. There is evidence that aging is one of the main
determinants of endothelial dysfunction in human vessels. The effect of aging can be detected
both in the micro and macrocirculation of forearm (Taddei, 1995a; Gerhard, 1996) and
coronary vessels (Vita, 1990; Yasue, 1990; Egashira, 1990; Zeiher, 1993) and is so strong
that in the forearm microcirculation its negative effect can be detected even in the presence
of essential hypertension (Taddei, 1995a). During exploration of the mechanisms responsible
for age-related endothelial dysfunction, it was observed that in normotensive subjects the
principle mechanism responsible for this alteration is a primary defect in the L-arginine-
NO pathway. In contrast, only in old subjects, around after 60 years, did EDCF production
starts to be detected and become relevant (Taddei, 1997a). In these older subjects, production
of cyclooxygenase-dependent factors is associated with a further and parallel impairment
in the L-arginine-NO pathway (Taddei, 1997a). If we consider essential hypertensive patients,
the mechanisms involved in age-related endothelial dysfunction are similar to those found
in healthy individuals, but characterized by an earlier onset. Therefore the production of
cyclooxygenase-dependent EDCF starts in an age range of 31–45 years, and in patients
older than 45 years the potentiating effect of indomethacin is augmented in parallel with
increasing age (Taddei, 1997a). Taken together, this series of results supports the possibility
that cyclooxygenase-dependent EDCF production is a phenomenon characteristic of aging,
with essential hypertension merely causing earlier onset of this endothelial alteration.
Figure 3.3 Acetylcholine-induced increase in forearm blood flow (FBF) in the absence (left) and presence
(right) of indomethacin (50 µg/100 ml forearm tissue/min) under control conditions (saline at 0.2 ml/min)
and in the presence of N G-monomethyl-L-arginine (L-NMMA, 100 µg/100 ml forearm tissue/min) in
essential hypertensive patients (n=7). Data are shown as means ±SD and expressed as absolute values. *
denotes a significant difference between infusion with and without L-NMMA (p<0.05 or less) (adapted
from Taddei, 1997a).
WHAT IS THE NATURE OF CYCLOOXYGENASE-DEPENDENT

EDCFS?
Experimental evidence suggests that cyclooxygenase-dependent EDCFs could be

prostanoids such as thromboxane A2 or prostaglandin H2 or oxygen free radicals (mainly
superoxide anions). It is significant that in essential hypertensive patients, EDCF
production is not the only endothelial alteration identified; rather it seems to be associated
with a simultaneous impairment in the L-arginine-NO pathway (Panza, 1993a; Panza,
1993b; Taddei, 1994; Taddei, 1995b). Thus vasodilation to acetylcholine is resistant to
L-arginine or L-NMMA induced facilitation or inhibition, respectively, suggesting the
presence of a decrease in NO availability in patients with essential hypertension. In these
patients, intrabrachial indomethacin not only increases the vasodilating response to
acetylcholine but also restores the facilitating and inhibiting effect of L-arginine and L-
NMMA (Figure 3.3) respectively, indicating that cyclooxygenase activity produces
substances which reduce NO availability (Taddei, 1997b). These substances are probably
oxygen free radicals since intrabrachial infusion of the scavenger vitamin C not only
increases the response to acetylcholine, but also restores the inhibiting effect of L-NMMA
(Taddei, 1998). Moreover, when simultaneously tested in the same study population,
68 S.Taddei et al.
Figure 3.4 Acetylcholine-induced increase in forearm blood flow (FBF) in the presence of saline (0.2 ml/
min); indomethacin (50 µg/100 ml forearm tissue/mm); vitamin C (8 mg/100 ml forearm tissue/min) and
simultaneous indomethacin and vitamin C (?) in essential hypertensive patients (n=7). Data are shown as
means±SD and expressed as absolute values. *denotes a significant difference between infusion in control
conditions and in the presence of vitamin C, indomethacin or vitamin C plus indomethacin (p<0.05 or less)
(adapted from Taddei, 1998).
vitamin C and indomethacin potentiate the response to acetylcholine to a similar degree

(Figure 3.4) and infusing both compounds together does not cause a further additional
improvement in endothelium-dependent vasodilation (Figure 3.4) (Taddei, 1998). Finally,
preliminary observations in aged (older than 60 years) normotensive subjects indicate
that vitamin C can improve vasodilation to acetylcholine, with an effect similar to that
observed with indomethacin.
Taken together these findings seem to indicate that, in ageing and essential hypertension,
EDCFs are likely to be oxygen free radicals produced by cyclooxygenase activity. However,
whether these oxidative substances are directly produced by cyclooxygenase or indirectly
by thromboxane A2 or prostaglandin H2 activity is still to be established. Future experiments
with specific inhibitors of thromboxane synthase or selective antagonists for thromboxane
A2 or prostaglandin H2 receptors, would be necessary to determine the contribution of these
substances.
Until now, cyclooxygenase-dependent EDCF production has been identified only during
stimulation of the endothelium by acetylcholine, and there are no data for other specific
receptor operated endothelial agonists such as bradykinin, substance P, serotonin or
endothelial responses activated by physical stimuli such as flow increase in large arteries
(see chapter 10).
Furthermore, cyclooxygenase-derived vasoconstrictor factors are not produced in baseline
conditions and do not modulate tonic NO release. Thus intrabrachial L-NMMA injection
causes a dose-dependent vasoconstriction, which is related to basal NO production (Vallance,
1989). In essential hypertension the vascular response to L-NMMA is reduced, indicating
a decrease in basal NO release (Calver, 1992; Taddei, 1995b). However, when L-NMMA is
tested in the presence of indomethacin in the forearm circulation of essential hypertensive
patients, cyclooxygenase inhibition does not improve the blunted vasoconstrictor response
to the NO-synthase inhibitor, demonstrating that EDCF production is not responsible for
the impaired basal release of NO (Taddei, 1997b). Vitamin C is also devoid of effect on
vasoconstrictor response to L-NMMA in essential hypertension, making it unlikely that
oxidative stress contributes to this impairment (Taddei, 1998).
Thus although patients with essential hypertension are characterized by a dysfunction in
basal NO-mediated dilatation and receptor-operated endothelium-dependent relaxation, the
mechanisms responsible for these defects are profoundly different, with the involvement of
cyclooxygenase activity only in the latter.
OTHER CLINICAL CONDITIONS CHARACTERIZED BY

CYCLOOXYGENASE-DEPENDENT EDCFs: ACUTE ESTROGEN
DEFICENCY AND HEART FAILURE
Acute endogenous estrogen deprivation is a clinical condition characteristic of women who

undergo ovariectomy. Estrogen can protect the vessel wall not only by improving lipid
profile (Bush, 1987), but also by acting on endothelial responses (Giscard, 1988; Williams,
1990). Thus the menopause is characterized by endothelial dysfunction in both normotensive
and hypertensive females (Celermajer, 1994; Taddei, 1996b).
Normotensive women who undergo ovariectomy and hysterectomy provide a good model
to evaluate the role of acute estrogen in modulating endothelial responses. In these subjects
ovariectomy was associated with a decrease in response to acetylcholine, but not to sodium
nitroprusside (Pinto, 1997). This phenomenon was detected within one month of surgery and
normal endothelium-dependent vasodilation was restored by estrogen replacement therapy
(ERT) (Pinto, 1997). These results confirm that estrogen can preserve endothelial function. In
line with this interpretation, prior to ovariectomy indomethacin did not change the response
to acetylcholine while L-NMMA was able to blunt it indicating that in these subjects,
cyclooxygenase-dependent EDCFs are not produced and NO availability is maintained, as
expected in a population of normotensive women aged around 45 years. After ovariectomy,
indomethacin-induced facilitation of the response to acetylcholine was observed, while the
inhibiting effect of L-NMMA disappeared, suggesting that acute endogenous estrogen
deprivation leads to EDCF production and a parallel drastic decrease in NO availability. When
endothelial responses were tested again after ERT, vasodilation to acetylcholine was sensitive
to L-NMMA and no longer affected by indomethacin. EDCFs produced after estrogen
deprivation are probably oxygen free radicals since vitamin C, while ineffective on the
vasodilation to acetylcholine before ovariectomy, significantly increased the response to the
70 S.Taddei et al.
muscarinic agonist after ovariectomy and was again ineffective after ERT administration.
Taken together and in line with experimental evidence (Ghin, 1992), these results suggest
that estrogen protects endothelial function by inhibiting the production of cyclooxygenase-
dependent EDCFs, which are very likely to be oxygen free radicals.
Finally, in patients with congestive heart failure (New York Heart Association functional
class II-III), systemic cyclooxygenase inhibition with oral indomethacin (50 mg) induced a
slight (+39%), but statistically significant, increase in the response to acetylcholine (Katz,
1993). However no information is available on the characterization of the cyclooxygenase-
dependent vasoconstrictor substances released in response to acetylcholine in patients with
heart failure.
CONCLUSIONS
Endothelial cells are capable of producing contracting factors including cyclooxygenase

derivatives. While production of these substances is characteristic of aging, essential, but
not secondary, hypertension, causes an acceleration and enhancement of the change.
Cyclooxygenase-dependent EDCFs are also produced in acute estrogen deprivation and in
heart failure. At least in the peripheral circulation, the nature of these substances seems to
be mainly related to oxidative stress, which reduces NO availability. Since in physiological
conditions NO has a protective effect on the vessel wall by inducing constant vasodilation,
inhibition of platelet aggregation, smooth muscle cell proliferation, monocyte adhesion
and endothelin-1 production, it is conceivable that in pathological conditions production of
cyclooxygenase-dependent EDCFs can lead to impaired NO effectiveness, thereby
diminishing the protective role of endothelial cells (Taddei, 1997c). However, the
mechanisms that regulate the balance between relaxing and contracting factors and the
process through which the endothelium loses its protective function by becoming a source
of substances such as cyclooxygenase-dependent EDCFs with vasoconstrictor,
proaggregatory and promitogenic activity remain to be determined.
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Endothelium-dependent relaxations cannot be fully explained by the release of either NO or/and prostacyclin.
Another unidentified substance(s) which hyperpolarises the underlying vascular smooth muscle cells, termed
endothelium-derived hyperpolarizing factor (EDHF), may contribute to endothelium-dependent relaxations,
especially in small arteries. In human blood vessels, endothelium-dependent hyperpolarisations are observed in
those blood vessels which exhibit endothelium-dependent relaxations that are partially or totally resistant to
inhibitors of NO synthase and cyclooxygenase. The existence of EDHF as a diffusable substance has been
demonstrated with isolated animal blood vessels but also with cultured human umbilical vein endothelial cells.
Hyperpolarisations caused by EDHF are insensitive to glibenclamide and iberiotoxin but are inhibited by
tetraethylammonium or the combination of charybdotoxin plus apamin or apamin plus ciclazindol. This indicates
that EDHF does not activate ATP-sensitive or large conductance calcium-activated potassium channels but rather
an undetermined population of potassium channels which may contain a subunit of small conductance calcium-
activated potassium channels. The identification of EDHF is unresolved and the suggestion that it could be a
cytochrome P450 metabolite of arachidonic acid is still controversial. The identification of EDHF and/or the
discovery of specific inhibitors of its synthesis and its action will allow a better understanding of its physiological
and pathophysiological role(s).
Key words: EDHF, endothelium, smooth muscle, potassium channels, membrane potential, vasodilatation.
4 Endothelium-Derived Hyperpolarizing Factor
Michel Félétou and Paul M.Vanhoutte
Institut de Recherches Internationale Servier, 6 Place des Pleides, 92415 Courbevoie,

Cedex, France
INTRODUCTION
Endothelial cells synthesise and release vasoactive mediators in response to various

neurohumoral substances (e.g. acetylcholine, adenosine trisphosphate, bradykinin, thrombin,
etc.) and physical stimuli (e.g. shear stress). Nitric oxide (NO) produced from the L-arginine
by endothelial NO synthase and prostacyclin produced from arachidonic acid by
cyclooxygenase have been identified as endothelium-derived vasodilators. However, not
all endothelium-dependent relaxations can be fully explained by the release of either NO
and/or prostacyclin. Another unidentified substance(s) which hyperpolarizes the underlying
vascular smooth muscle cells, termed endothelium-derived hyperpolarizing factor (EDHF),
may contribute to endothelium-dependent relaxations (for review see Furchgott and
Vanhoutte, 1989, Félétou and Vanhoutte, 1996, Mombouli and Vanhoutte, 1997).
EXISTENCE OF AN ALTERNATIVE PATHWAY
Besides prostacyclin and NO [the endothelium-derived relaxing factor discovered by

Furchgott and Zawadzki (1980)], the existence of a third mediator was postulated by DeMey
et al. in 1982. In 1984, Bolton et al. demonstrated that a muscarinic agonist elicited
endothelium-dependent hyperpolarization of vascular smooth muscle cells. This
phenomenon has been confirmed in various blood vessels from different species (Félétou
and Vanhoutte, 1985, 1988; Komori and Suzuki, 1987; Taylor et al., 1988; Chen et al.,
1988; Keef and Bowen, 1989; Brayden, 1990). When L-arginine analogues became available
as specific inhibitors of the production of NO (Rees et al., 1989), it soon appeared that
endothelium-dependent relaxations and hyperpolarizations could be partially or totally
resistant to inhibitors of cyclooxygenase and NO synthase (Bény and Brunet, 1988a & b;
Huang et al., 1988; Richard et al., 1990; Cowan and Cohen, 1991; Mugge et al., 1991;
Hasunuma et al., 1991; Illiano et al., 1992; Nagao et al., 1992a; Nagao and Vanhoutte,
1992b; Suzuki et al., 1992), suggesting the existence of an additional endothelial mechanism.
Furthermore, endothelium-dependent responses, which are resistant to inhibitors of NO
synthase and cyclooxygenase, are observed without an increase in intracellular levels of
cyclic nucleotides (cyclic GMP and cyclic AMP) in the smooth muscle cells (Taylor et al.,
1988; Cowan and Cohen, 1991; Mombouli et al., 1992; Zygmunt et al., 1994; Garcia-
Pascual et al., 1995). Under these conditions, the hyperpolarization of the cell membrane
of vascular smooth muscle and the resulting reduction in Ca2+ entry explains the endothelium-
dependent relaxations (Nagao and Vanhoutte, 1991, 1992a; Garland and McPherson, 1992).
Indeed, hyperpolarization of smooth muscle cells reduces the open probability of
75
76 M.Félétou and P.M.Vanhoutte
voltage-dependent calcium channels, thereby decreasing calcium influx and lowering

intracellular calcium levels (Nelson et al., 1990). In addition, the hyperpolarization may
reduce the increase in intracellular phosphatidylinositol turnover caused by agonist-induced
receptor activation (Itoh et al., 1992).
NO can activate various potassium conductance: ATP-sensitive potassium channels
(K-ATP) via a cyclic GMP-dependent mechanism (Miyoshi et al., 1994) and large
conductance calcium-activated potassium channels (BKCa) either via a cyclic GMP-
dependent mechanism (Robertson et al., 1993; Taniguchi et al., 1993; Archer et al., 1994)
or even directly (Bolotina et al., 1994; Shin et al., 1997). Hence NO could therefore
produce hyperpolarization of the smooth muscle cells. Indeed, in blood vessels from
species such as rat, guinea-pig and rabbit, endothelium-dependent hyperpolarizations
resistant to inhibitors of NO synthase and cyclooxygenase and hyperpolarizations to
endothelium-derived or exogenous NO can be observed in the same vascular tissue (Tare
et al., 1990; Murphy and Brayden, 1995a; Parkington et al., 1995; Plane et al., 1995;
Corriu et al., 1996b). Likewise, prostacyclin and its stable analogue, iloprost, also can
hyperpolarize vascular smooth muscle cells (Siegel et al., 1987; Parkington et al., 1993;
Corriu et al., 1996b). Thus incomplete inhibition of the NO-synthase and/or
cyclooxygenase could account for endothelium-dependent hyperpolarization (Cohen et
al., 1997). However, in blood vessels such as the carotid artery of the guinea-pig the
combination of NO scavengers and inhibitors of nitric oxide synthase (in the presence of
inhibitor of cyclooxygenase) does not inhibit endothelium-dependent hyperpolarizations
(Figure 4.1). Finally, in canine and porcine blood vessels hyperpolarization cannot be
produced by nitrovasodilators or exogenous NO (Bény and Brunet, 1988a; Komori et al.,
1988; Pacicca et al., 1992) indicating that in most species a third pathway has to be
involved in order to fully explain endothelium-dependent relaxations.
Mechanism of Endothelium-Dependent Hyperpolarization

Endothelium-dependent hyperpolarization involves the opening of a potassium
conductance. The amplitude of the hyperpolarization is inversely related to the extracellular
concentration of K+ ions, and it disappears in K+ concentrations higher than 25 mM (Chen
and Suzuki, 1989; Nagao and Vanhoutte, 1992a; Corriu et al., 1996a). Furthermore,
endothelium-dependent hyperpolarizations are associated with an increase in rubidium
uptake (Taylor et al., 1988). Finally, non-selective potassium channel inhibitors, such as
tetraethylammonium (TEA) or tetrabutylammonium prevent the hyperpolarization (Chen
et al., 1991; Nagao and Vanhoutte, 1992a; Van de Voorde et al., 1992). The exact nature
of the potassium conductance involved is still unknown and may depend on the species.
In arteries of rats, rabbits, bovines and pigs, responses attributed to EDHF are blocked by
apamin suggesting, in these species, the involvement of calcium-dependent potassium
channels of small conductance (SKCa; Adeagbo and Triggle, 1993; Hecker et al., 1994;
Garcia-Pascual et al., 1995; Murphy and Brayden, 1995b). In rat mesenteric and guineapig
carotid arteries, the combination of charybdotoxin (an inhibitor of both BKCa and voltage-
sensitive potassium channels) plus apamin is required to abolish the hyperpolarization
resistant to inhibitors of NO synthase and cyclooxygenase (Figure 4.2) while each blocker
individually is either ineffective (charybdotoxin) or only partially effective (apamin)
(Garland and Plane, 1996; Corriu et al., 1996b; Chataigneau et al., 1998a). The
combination of iberiotoxin (a specific inhibitor of BKCa) plus apamin did not mimic the effects
Endothelium-Derived Hyperpolarizing Factor 77
Figure 4.1 Membrane potential recording in guinea-pig isolated carotid artery with endothelium.
* First trace from the top: Acetylcholine (ACh, 1 µM) induced hyperpolarization in the presence of an inhibitor of
nitric oxide synthase L-nitro-arginine (L-NA, 100 µM) and an inhibitor of cyclooxygenase (indomethacin, 5 µM).
The hyperpolarization was not observed in preparations without endothelium (not shown).
* Second trace from the top: Acetylcholine in the presence of L-NA plus indomethacin plus a second inhibitor of
nitric oxide synthase L-nirromonomethyl-arginine (L-NMMA, 100 µM) plus a scavenger of nitric oxide
(haemoglobin, 10 µM) still induced endothelium-dependent hyperpolarization.
* Third trace from the top: Acetylcholine in the presence of L-NA plus indomethacin plus a different scavenger of
nitric oxide 2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-loxyl-3-oxide (carboxy-PTIO, 10 µM) still
induced endothelium-dependent hyperpolarization.
* Fourth trace from the top: Acetylcholine in presence of L-NA plus indomethacin plus carboxy-PTIO plus a
second inhibitor of cyclooxygenase (meclofenamic acid, 10 µM) still induced endothelium-dependent
hyperpolarization.
of charybdotoxin and apamin (Zygmunt and Högestätt, 1996; Chataigneau et al., 1998a)
indicating that BK Ca channels are not involved in the endothelium-dependent
hyperpolarization of the guinea-pig carotid and rat mesenteric artery. In contrast, in the
rabbit carotid artery relaxations attributed to EDHF are sensitive to charybdotoxin alone
(Cowan et al., 1993). A unifying hypothesis once proposed by Zygmunt et al. (1997a)
suggested that the potassium channel(s) involved in the endothelium-dependent
hyperpolarization was either a “classical” type of SKCa or a channel similar to one of the
apamin-insensitive variants of SKCa, some of which are sensitive to charybdotoxin alone
Figure 4.2 Effect of acetylcholine (ACh, 1 µM, upper traces), S-nitroso-L-glutathione (a nitrovasodilator, 10 µM,
middle traces) and iloprost (a stable analogue of prostacyclin, 0.1 µM, lower traces) in the guinea-pig isolated
carotid artery with endothelium
Left panel: control experiments in presence of L-nitro-arginine (L-NOARG, 100 µM) and indomethacin (5µM).
Middle panel: treatment with glibenclamide (1 µM).
Right panel: treatment with the combination of charybdotoxin (0.1 µM) plus apamin (0.5 µM).
Bar graphs summarised the changes in membrane potential obtained in the various experimental conditions. The
effects of S-nitroso-L-glutathione were mimicked by two other nitrovasodilators: sodium nirroprusside (SNP, 10
µM) and SIN-1 (10 µM).
Modified from Corriu et al. (1996b).
(Van Renterghem and Lazdunski, 1992; Köhler et al., 1996). It has been assumed that the
targets of potassium channel blockers were situated on the vascular smooth muscle cells
because these toxins inhibited EDHF-mediated responses without affecting the relaxations
or the hyperpolarizations produced by endothelial nitric oxide or prostacyclin e.g. being
selective (Corriu et al., 1996b; Zygmunt and Höggestätt, 1996; Petersson et al., 1997;
Chataigneau et al., 1998a; Zygmunt et al., 1998). However, calcium-activated potassium
channels are also expressed in endothelial cells (Marchenko and Sage, 1996). In the
endothelial cells of the guinea-pig coronary artery, the combination of the two toxins does
not affect the acetycholine-induced increase in intracellular free calcium concentration
(Yamanaka et al., 1998). However, in the hepatic artery of the rat and in the aortic valve of
the rabbit, the combination of charybdotoxin plus apamin inhibits the hyperpolarization of
the endothelial cells produced by acerylcholine (Edwards et al., 1998; Ohashi et al., 1999).
Thus, charybdotoxin and apamin can act on the endothelial cells and this endothelial effect
could be responsible for the inhibition of the EDHF-mediated responses. Therefore, the
vascular smooth muscle population of potassium channel activated during the endothelium-
dependent hyperpolarization is still unknown.
The sustained membrane hyperpolarizations provoked by prostacyclin (and iloprost)
are blocked by glibenclamide, suggesting the involvement of ATP-dependent potassium
channels (Parkington et al., 1993, 1995; Corriu et al., 1996b; Figure 4.2). Likewise, in
blood vessels where NO causes hyperpolarization, as recorded with intracellular
microelectrodes, the response is also blocked by glibenclamide (Miyoshi et al., 1994; Plane
et al., 1995; Murphy and Brayden, 1995a; Parkington et al., 1995; Corriu et al., 1996b) but
not by the combination of charybdotoxin plus apamin (Corriu et al., 1996b, Figure 4.2). By
contrast, glibenclamide does not inhibit endothelium-dependent hyperpolarization resistant
to inhibitors of NO synthase and cyclooxygenase confirming the existence of a third pathway
(Chen et al., 1991; McPherson and Angus, 1991; Fujii et al., 1992; Nakashima et al., 1992;
Nagao and Vanhoutte, 1992a; Plane et al., 1995; Corriu et al., 1996b; Figure 4.2).
Nature of EDHF
The term EDHF implicitly refers to a factor released by the endothelial cells. However,
theoretically, endothelium-dependent hyperpolarization could involve electrical coupling
through myo-endothelial junctions. Indeed, substances which produce endothelium-
dependent hyperpolarization of vascular smooth muscle cells, also hyperpolarize endothelial
cells, with the same time course (Busse et al., 1988; Davies et al., 1988; Brunet and Beny,
1989; Colden-Stanfield et al., 1990; Chen and Cheung, 1992). However, if electrical coupling
from smooth muscle to endothelial cells exists, electrical propagation in the reverse direction
does not seem to occur (Marchenko and Sage, 1993; Bény and Gribi, 1989; Bény and
Pacicca, 1994). Dye studies demonstrate that couplings between endothelial and smooth
muscle cells do not necessarily occur (Beny, 1990). Even in tissues where heterocellular
dye coupling between endothelial and smooth muscle cells is observed, hyperpolarization
of endothelial cells is not propagated to the smooth muscle cells (Beny et al., 1997).
Furthermore, halothane or heptanol, agents which uncouple cells linked by gap junctions,
do not inhibit endothelium-dependent hyperpolarizations (Bény and Pacicca, 1994; Bény
and Chabaud, 1996; Zygmunt and Högestätt, 1996).
However, more specific blockers of gap junctions, such as 18-glycyrrhetinic acid and
Gap27 (a peptide which possesses a conserved sequence homology with a portion of
connexin) inhibit EDHF-like responses in rabbit and guinea-pig arteries (Chaytor et al.,
1998; Yamamoto et al., 1998; Taylor et al., 1998; Yamamoto et al., 1999). At present, this
mechanism needs to be further explored to better understand its potential contribution to
EDHF-mediated responses. Gap junctions could be the site of myo-endothelial electrical
cell coupling but could also be the site of transfer of low molecular weight compounds.
Furthermore, the role of gap junctions between the innermost intimal vascular smooth muscle
cells and the deeper layers has also to be clarified.
Thus, endothelium-dependent hyperpolarizations resistant to inhibitors of NO synthase
and cyclooxygenase inhibition have been attributed to the release of a yet unidentified
diffusable substance (EDHF), the existence of which has been demonstrated under bioassay
conditions whereby the source of EDHF was either native vascular segments or cultured
endothelial cells (Félétou and Vanhoutte, 1988; Kauser et al., 1989; Chen et al., 1991;
Mombouli et al., 1996; Popp et al., 1996a; Harder et al., 1996; Fukuta et al., 1996). The
technical difficulties in demonstrating the diffusable nature of EDHF can be explained
by a very short half-life of the substance, its preferential abluminal release (Kauser et al.,
1992) the simultaneous release of a hypothetical endothelium-derived depolarizing factor
(Mombouli et al., 1996; Corriu et al., 1996b) or a combination of these. The release of
EDHF requires an increase in endothelial intracellular calcium and the subsequent
Figure 4.3 Membrane potential of vascular smooth muscle cells in human isolated coronary artery with endothelium
in presence of L-nitro-arginine (100 µM) and indomethacin (10 µM) and perindoprilat (1 µM). Effects of
glibenclamide on the changes in membrane potential produced by lemakalim (1 µM, A), bradykinin (10 nM, B)
an calcium ionophore A 23187 (1 µM, C) on membrane potential. Tissues in A and C were obtained from the heart
of a 49 year old alcoholic cardiomyopathy and B from a 62 year old idiopathic cardiomyopathy. From Nakashima
et al. (1993, with the permission of the American Heart Association).
activation of calmodulin (Chen and Suzuki, 1990, Nagao et al., 1992b, Illiano et al.,
1992). The calcium and calmodulin dependency of responses mediated by EDHF is similar
to that observed with endothelial NO-dependent relaxation although the former appears
to be more sensitive to calmodulin blockers than the latter (Bredt and Snyder, 1990;
Illiano et al., 1992).
It has been suggested that EDHF may be a short-lived metabolite of arachidonic acid
produced through the cytochrome P450 monooxygenase pathway (Rubanyi and Vanhoutte,
1987, Komori and Vanhoutte, 1990). Inhibitors of this pathway inhibit endothelium-
dependent vasodilator responses resistant to inhibitors of NO synthase and cyclooxygenase
in the perfused heart and kidney of the rat and in isolated porcine and bovine coronary
arteries (Bauersachs et al., 1994; Hecker et al., 1994; Fulton et al., 1992 and, 1995).
Some metabolites of arachidonic acid formed by the cytochrome P450 activate K+ channels
in vascular smooth muscle cells (Gebremedhin et al., 1992, Hu and Kim, 1993). Muscarinic
agonists induce not only endothelium-dependent relaxation and hyperpolarization of
bovine coronary arterial smooth muscle but also the release of epoxyeicosatrienoic acids
from bovine coronary arterial endothelial cells. These responses are inhibited by SKF
525A and miconazole (Campbell et al., 1996). The cytochrome P450 metabolites,
produced by the endothelial cells, increase the open-state probability of calcium-activated
potassium channels sensitive to TEA or charybdotoxin, and induce hyperpolarization of
coronary arterial smooth muscle cells. Taken in conjonction these observations support
the hypothesis that epoxyeicosatrienoic acids could be EDHF. However, cytochrome P450
inhibitors studied at high concentrations, are notoriously unspecific and can inhibit
hyperpolarizations induced by potassium channel openers such as levcromakalim (Graier
et al., 1995a; Eckman et al., 1995; Edwards et al., 1996; Vanhoutte and Félétou, 1996).
In blood vessels of rats, guinea-pigs, dogs and pigs, chemically unrelated inhibitors of
cytochrome P450 do not inhibit the EDHF responses or produce a non specific inhibition
(Corriu et al., 1996a & c; Graier et al., 1996; Zygmunt et al., 1996; Fukao et al., 1997;
Chataigneau et al., 1998a).
Randall et al. (1996) postulated that anandamide is EDHF. This arachidonic acid derivative
is an endogenous ligand for the cannabinoid CB1 receptor (Devane et al., 1992; Di Marzo
et al., 1994). Anandamide induces dilatation which mimics responses to EDHF in the isolated
and perfused mesenteric and coronary arterial bed of the rat (Randall et al., 1997; Randall
and Kendall, 1997 and 1998). However, in the kidney dilatation caused by anandamide is
due to the release of NO (Deutsch et al., 1997). In isolated blood vessels from various
species (pig, guinea-pig, rat) anandamide does not produce hyperpolarization or if it does
so the underlying mechanism differs from EDHF-mediated responses. Indeed some of these
responses to anandamide are endothelium-dependent (Zygmunt et al., 1997b, Chataigneau
et al. 1998b). Finally CB1 receptor antagonists do not inhibit endothelium-dependent
hyperpolarization. These observations do not support the proposal that an endogenous
cannabinoid is the major mediator of endothelium-dependent hyperpolarizations (Campbell
et al., 1997; Chataigneau et al., 1997b, 1998b; Plane et al., 1997; Zygmunt et al., 1997;
White and Hiley, 1997,).
In the rat hepatic artery, potassium ion could be EDHF (Edwards et al., 1998). Indeed,
endothelial cells hyperpolarize in response to neurohumoral substances which produce the
release of vasoactive substances such as NO and EDHF. Potassium ions, flowing through
the opening of endothelial small and intermediate conductance potassium channels (sensitive
to charybdotoxin and apamin), may accumulate in the intercellular space. This rise in
potassium concentration could hyperpolarize the smooth muscle by activating the inward
rectifying potassium channels (sensitive to low concentrations of barium) and the sodium/
potassium pump (sensitive to ouabain). However, in other blood vessels the addition of
potassium does not necessarily produce hyperpolarization, possibly because the inward
rectifying potassium channel is expressed poorly in certain vascular smooth muscle cells
especially in those large blood vessels. Furthermore, in the guinea-pig carotid and porcine
coronary arteries, the endothelium-dependent hyperpolarization is not affected by the
combination of ouabain and barium (Quignard et al., 1999). Further studies are required to
verify the pertinence of this proposal and to determine the vascular beds where it applies
(Vanhoutte, 1998).
Molecules such as carbon monoxide, hydroxyl radicals, hydrogen peroxide all are putative
EDHF as they are produced by the endothelial cells and induce hyperpolarisation of the
smooth muscle cells. However, the evidence conforting the role of these molecules as EDHF
is either weak or inexistent (For review see Mombouli and Vanhoutte, 1997). It has been
recently suggested that different pathways could be involved in the EDHF responses. Thus,
in the rabbit femoral artery, the relaxation attributed to EDHF could involve the activation
of both heme-oxygenase-and cannabinoid CB1 receptor antagonist-sensitive pathways (Rowe
et al., 1998). This proposition should be confirmed and explored in other vascular beds. At
present, from the data available it is not possible to conclude that EDHF has been identified
with certainty.
ENDOTHELIUM-DERIVED HYPERPOLARIZING FACTOR IN

HUMANS
Studies which have specifically addressed EDHF-mediated responses in human blood vessels
are scarce. The earliest electrophysiological evidence of an endothelium-dependent
hyperpolarization in human blood vessels was obtained in isolated coronary arteries
(Nakashima et al., 1993). Bradykinin and the calcium ionophore A23187 induced a transient
hyperpolarization which was resistant to inhibitors of nitric oxide synthase and
cyclooxygenase, indicating the involvement of EDHF (Figure 4.3). This hyperpolarization
was associated with a corresponding endothelium-dependent relaxation resistant to the same
inhibitors. These earlier results in the human coronary artery have been confirmed (He,
1997a and b). Similarly, endothelium-dependent hyperpolarizations, associated with
endothelium-dependent relaxation resistant to inhibitors of nitric oxide synthase and
cyclooxygenase have been observed in human pial arteries in response to substance P
(Petersson et al., 1995) and in gastroepiploic arteries of different sizes in response to
bradykinin and to a lesser extent to acetylcholine (Urakami-Harasawa et al., 1997).
Acetylcholine also induces endothelium-dependent hyperpolarizations in the human
saphenous vein in presence of nitric oxide synthase and cyclooxygenase inhibitors (Yang
and He, 1997).
Evidence for the presence of a functional role of EDHF comes from isolated human
arteries in which endothelium-dependent relaxations resistant to inhibitors of nitric oxide
synthase and cyclooxygenase were observed. This phenomenon has been reported in
coronary (Nakashima et al., 1993; Stork and Cocks, 1994; Kemp and Cocks, 1997; He,
1997a and b), subcutaneous (Woolfson and Poston, 1991; Deng et al., 1995; Van de Voorde
et al., 1997), omental (Pascoal and Umans, 1996; Ohlmann et al., 1997; Wallerstedt and
Bodelsson, 1997), renal (Kessler et al., 1996) and radial arteries (Hamilton et al., 1997).
By contrast, EDHF-dependent responses are small or inexistent in the internal thoracic
artery (Hamilton et al., 1997) and in the basilar artery (Hatake et al., 1990).
As in animal arteries (Mügge et al., 1991; Nagao et al., 1992a; Shimokawa et al., 1996),
the contribution of the EDHF response is significantly greater in small than in large human
arteries (Woolfson and Poston, 1990; Urakami-Harasawa et al., 1997).
It has not been possible yet to evaluate, in the intact human, the involvement of EDHF in
the vasodilator responses to various stimuli as specific inhibitors of its production or its
action are not available. An EDHF mechanism is often suggested to explain vasodilatation
resistant to inhibitors of nitric oxide synthase. However, numerous other interpretations are
possible. First of all, most of the human studies do not involve the administration of an
inhibitor of cyclooxygenase. Furthermore, complete blockade of nitric oxide synthase is
difficult to obtain, and/or the non-endothelial effect of the vasodilators such as a direct
effect on the smooth muscle cells or an inhibitory effect on the sympathetic nerve endings
cannot be excluded easily. Therefore, the exact role of EDHF in the control of human blood
vessel tone is still unknown.
Mechanism of Endothelium-Dependent Hyperpolarisation in Human Vascular

Tissue
The EDHF-mediated responses (hyperpolarizations or/and relaxations resistant to inhibitors

of nitric oxide synthase and cyclooxygenase) can be attributed to the activation of potassium
channels as they are blocked by elevated potassium concentrations. These responses are
independent of K-ATP activation (Figure 4.3) and are minimally or not at all affected by
charybdotoxin, iberiotoxin or apamin individually but are blocked either by non-specific
potassium channel inhibitors (tetrabutylammonium or tetraethylammonium) or by the
combination of charybdotoxin plus apamin (Nakashima et al., 1993; Kessler et al., 1996;
Pascoal and Umans, 1996; Ohlmann et al., 1997; Petersson et al., 1997; Urakami-Harasawa
et al., 1997; Wallerstedt and Bodelsonn, 1997). In small arteries dissected from gluteal fat,
the partial inhibition observed with tolbutamide, a K-ATP inhibitor, is probably due to a
non specific effect of the high concentration of the compound (Deng et al., 1995). In the
human hand vein, vasodilatations to substance P are partially resistant to inhibitors of nitric
oxide synthase and cyclooxygenase but are partially inhibited by the sulfonylurea glyburide.
Again, this effect may not be related to an inhibition of the EDHF pathway. Indeed, in the
human vein the effect of substance P involves the activation of the cyclooxygenase pathway
without apparent involvement of NO synthase. The effects of glyburide and acetylsalicylic
acid are not additive suggesting that K-ATP could have been activated by the products of
cyclooxygenase (Strobel et al., 1996). Although the potassium conductance involved in the
endothelium-dependent hyperpolarization in human blood vessels is not identified with
precision, these limited data available are consistent with observations already made in
other species.
Nature of EDHF in Human Arteries

Human endothelial cells hyperpolarize in response to mediators which induce endothelium-
dependent responses (Groschner et al., 1994; Ochi et al., 1995). Since no data seem available
concerning electrical coupling through myo-endothelial junctions in human blood vessels
this phenomenon cannot be ruled out. However, a diffusable hyperpolarizing factor released
from cultured human umbilical vein endothelial cells has been detected in the presence of
combined treatment with inhibitors of NO-synthase and cyclooxygenase (Popp et al., 1996b),
demonstrating the production of an EDHF by human endothelial cells.
In intact human arteries, the effect of endogenous NO (or nitrovasodilators) on cell
membrane potential is unknown. In cultured human pulmonary vascular smooth muscle
cells, NO activates calcium-dependent potassium channels through a cyclic GMP-dependent
mechanism (Peng et al., 1996). This may indicate that NO provokes hyperpolarization of
vascular smooth muscle in the pulmonary circulation. An incomplete inhibition of the NO-
synthase by the inhibitors could explain the endothelium-dependent hyperpolarisation.
However, this interpretation is unlikely at least in coronary arteries. In the presence of the
combination of inhibitors of NO-synthase and cyclooxygenase plus oxyhemoglobin which
scavanges NO, a component of the bradykinin-induced endothelium-dependent relaxation
Figure 4.4 Membrane potential of vascular smooth muscle cells in human isolated coronary artery with (bottom
traces) and without endothelium (top traces) in presence of L-nitro-arginine (100 µM) and indomethacin (10 µM).
Effect of bradykinin in rings with and without endothelium. Tissues were obtained from the heart of a 15 month
old (A, congenital heart disease) and from a 64 year old (B, ischemic heart disease). From Nakashima et al.
(1996, by permission of Harwood Academic Publishers)
is still observed. This component is abolished by elevation of the extracellular potassium

concentration (Kemp and Cocks, 1997).
The involvement of a cytochrome P450 metabolite of arachidonic acid has been ruled
out in human coronary and omental arteries as quinacrine, proadifen and clotrimazole have
no effect on the EDHF responses (Ohlmann et al., 1997; Urakami-Harasawa et al., 1997;
Wallerstedt and Bodelsonn, 1997). In renal arteries the inhibitory effect of two anaesthetics,
etomidate and thiopental, has been considered to be an indication of the involvement of
cytochrome P450 (Kessler et al., 1996). In cultured endothelial cells from the human
umbilical vein a transferable ß-naphtoflavone-inducible hyperpolarizing factor is synthesised
(Popp et al., 1996b). However, even without considering the non-specific effects of
cytochrome P450 inhibitors already mentioned, activation of cytochrome P450 in human
endothelial cells appears to be a more general requirement for increasing the intracellular
calcium concentration and thus the release of endothelial derived factors such as NO and
EDHF (Graier et al., 1995b). This fundamental endothelial function of cytochrome P450
may confuse the issue when interpreting results of studies investigating the effects of
inhibitors of cytochrome P450 on EDHF-mediated responses.
CONCLUSION
Besides the release of NO and prostacyclin, a third endothelial pathway involving

hyperpolarization of the underlying vascular smooth muscle cells, contributes to the
Figure 4.5 Conclusion: Existence of Multiple EDHF(s)?

M3, B2, NK1: M3, muscarinic, B2 bradykinin and NK1 neurokinin receptor subtypes respectively; R: receptor;
A23187: calcium ionophore; NOS: nitric oxide synthase; COX: cyclooxygenase; cyt P450: cytochrome P450
monooxygenase; NO: nitric oxide; PGI2: prostacyclin; EDHF endothelium-derived hyperpolarizing factor; EDDF:
endothelium-derived depolarizing factor; 5,6 EET: 5,6-epoxy-eicosatrienoic acid; 11,12 EET: 11,12-
epoxyeicosatrienoic acid; 14,15 EET: 14,15-epoxy-eicosatrienoic acid; GC: guanylate cyclase, cGMP: cyclic
guanosine monophosphate; cAMP: cyclic adenosinesine monophosphate; ATP: adenosine trisphosphate; IP3: inositol
trisphosphate; TEA: tetraethyl ammonium; TBA: tetrabutyl ammonium, SR 141716: an antagonist of cannabinoid
CB1 receptor. The term EDHF should be restricted to describe phenomena in which endothelium-dependent
hyperpolarizations are insensitive to glibenclamide and resistant to inhibitors of nitric oxide synthase and
cyclooxygenase. Modified from Vanhoutte and Félétou (1996).
endothelium-dependent relaxation of various human blood vessels. Studies with animal

blood vessels show that this phenomenon is due to a diffusible factor, termed endothelium-
derived hyperpolarizing factor (EDHF), activating potassium channels. The identity of EDHF
and the exact nature of the potassium channel population activated by it remain to be
determined. In the absence of selective inhibitors of the synthesis or the action of EDHF, its
role cannot be evaluated fully in humans. The impairment of endothelium-dependent

hyperpolarization by ageing (Figure 4.4) or hypercholesterolemia and conversely the
enhancement of the responses attributed to EDHF by therapeutic agents such as converting
enzyme inhibitors or possibly estrogens, may indicate a pathophysiological role of this
third endothelial pathway (Nakashima et al., 1993; Urakami-Harasawa et al., 1997; Tagawa
et al., 1997), (Figure 4.5).
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Vascular haemostasis defined as the ability of the vascular system to preserve blood fluidity and vascular integrity
is maintained by the reciprocal interactions between the endothelium and blood cells. In physiology, platelets
remain in a close vicinity of the endothelial lining, an arrangement supported by central position of erythrocytes
in the blood stream. An accidental breach of the endothelial integrity triggers off homeostatic reactions aimed at
blood arresting and vessel wall repair. Platelets adhere to the place of injury and form haemostatic aggregate.
Platelet mediators such as adenosine diphosphate, thromboxane A2 and matrix metalloproteinase-2 mediate platelet
aggregation. The balancing effects of the inhibitor mediators including nitric oxide and prostacyclin prevent
excessive haemostatic reactions (thrombosis). Leukocytes participate in homeostatic endothelial responses, an
effect also regulated by the endothelial cells. Thus, the endothelial lining is the “maestro” of the vasculature
playing a key role in regulation of vascular haemostasis.
Key words: Platelets, leukocytes, erythrocytes, haemostasis, thrombosis, nitric oxide

5 Regulation of Blood Cell Function by Endothelial Cells
Marek W.Radomski and Anna S.Radomski
Division of Research, Lacer, SA, 08025 Barcelona, Spain and Department of

Pharmacology, University of Alberta, Edmonton, AB T6G 2H7, Canada
INTRODUCTION
Regulation of blood cell behaviour is a major endothelial function that ensures blood fluidity.
The endothelium actively supports the fluid state of flowing blood and prevents activation
of circulating cells. This ability must be carefully balanced against the homeostatic property
of blood that is meant to protect the vascular system from loss of blood due to an accidental
breach of vascular integrity. These diverse, yet co-ordinated, reactions are often referred to
as vascular haemostasis. Thrombosis is a pathological extension of haemostasis that takes
place when the regulatory mechanisms are inadequate.
The importance of the endothelial lining for preservation of blood fluidity is best
exemplified by the fact that artificial vessel prostheses as well as pathologically altered
endothelial cells are highly thrombogenic. In this chapter we will discuss the mechanisms
responsible for non-thrombogenic properties of vascular endothelium.
REGULATION OF PLATELET FUNCTION BY ENDOTHELIUM
Platelets
Blood platelets are small (approximately 2 µm in diameter) anucleate elements formed by
fragmentation of megakaryocytes. Non-activated (resting) platelets are discoid in shape
and contain numerous granules in the cytoplasm. The granules are composed of various
activating and proliferating agents whose function is crucial to platelet reactions (White
1988). Rheological studies have shown that pulsatile blood flow and the shear rate are the
major determinants of platelet behaviour in vivo (Slack, Cui and Turitto, 1993). The shear
stress is responsible for the tendency of suspended particles (blood elements) to move towards
the centre of the flowing stream. Erythrocytes are larger and more numerous than platelets
and tend to occupy the axial position in the blood stream forcing less numerous and smaller
platelets to remain in a close proximity of the endothelial lining.
The endothelial proximity of platelets results in generation of high shear forces acting
at the interface between platelet and the vessel wall. This assures, on one hand, prompt
and effective platelet recruitment to the place of accidental injury during the haemostatic
process, on the other hand, it allows tight endothelial control over the process of platelet
activation.
Correspondence: Marek W.Radomski, Departemnt of Pharmacology, University of Alberta, 9–50 MSB,

Edmonton T6G 2H7, Canada.
95
96 M.W.Radomski and A.S.Radomski
Platelet Activation
The biological signal for initiation of platelet activation is delivered by the exposure of
adhesive components of the subendothelium that are normally concealed from the blood
by the endothelium.
Platelet adhesion
Platelets make contact with adhesive proteins through specific adhesion receptors. In
vivo, under conditions of shear stress, binding of v. Willebrand factor to its receptors on
platelets and the counter receptors in the vessel wall, serves as a mechanism to anchor
platelets to the subendothelium (Ginsberg, Loftus and Plow, 1988). Following the initial
contact phase, platelets change their discoid shape to ameboid-like structures and spread
on the endothelium.
Platelet aggregation
The biological role of aggregation is to reinforce the platelet adhesion monolayer with a
structure based on web-like interactions between the adjacent platelets. The aggregate, thus
formed, is firm enough to withstand disintegrating stimuli brought about by blood flow and
shear forces (Figure 5.1).
The formation of aggregates requires dramatic rearrangements of platelet structure and
cytoskeleton and may be brought about by soluble activator agonists including thrombin,
adrenaline, serotonin and ADP. These factors trigger a biochemical cascade of events that
ultimately leads to the activation of the platelet integrin receptor IIb/IIIa and this allows
binding of fibrinogen to the receptors of adjacent platelets. The binding of fibrinogen results
in further reinforcement of the existing platelet plug (Radomski and Salas, 1995).
Platelet aggregation pathways
Dramatic reorganisation of platelet structure and formation of effective haemostatic plug is

brought about by activation of at least three metabolic pathways that amplify aggregation
and plug formation.
The first pathway is mediated by the biosynthesis from arachidonic acid of thromboxane
A2 (Hamberg, Svensson and Samuelsson, 1975). The generation of this eicosanoid is
inhibited by aspirin and aspirin-like drugs (Ferreira, Moncada and Vane, 1973), and this
property of aspirin is now widely used to decrease excessive platelet activation that is often
associated with vascular disorders including ischaemic heart disease and stroke (Patrono
and Renda, 1997).
The second pathway of aggregation is mediated by the release from platelet granules of
ADP that activates platelets via specific receptors (Born, 1985; Puri and Colman, 1997).
The pharmacological modulators of ADP-induced platelet aggregation include ticlopidine
and dipyridamole (Schafer, 1996).
We have recently investigated non-thromboxane, non-ADP mechanisms of platelet
aggregation and found that the release from platelets of matrix metalloproteinase-2 enzyme
(MMP-2) may mediate some of these reactions (Sawicki et al., 1997). Platelet activation
leads to the translocation of MMP-2 from the cytosol to the extracellular space. During the
Regulation of Blood Cell Function by Endothelial Cells 97
Figure 5.1 The physiology of platelet haemostasis.
early stages of aggregation MMP-2 remains in close association with the platelet plasma
membrane and this association is likely to stimulate platelet aggregation (Sawicki et al.,
1998) (Figure 5.2). Endogenous and pharmacological inhibitors of metalloproteinases inhibit
MMP-2-induced platelet aggregation.
Physiological Control of Platelet Activation

An inherent capacity of platelets to aggregate and “seal the rents” in the vascular system is
controlled by the systems that inhibit platelet activation. Under physiological conditions
the balance between the activator and inhibitor systems of platelet activation process is
Figure 5.2 Activation-induced translocation of platelet MMP-2.
tightly maintained, preserving the fluid state of blood, thus vascular haemostasis and
homeostasis. An imbalance between these groups of mediators may result in a disturbance
of haemostasis exemplified by bleeding diathesis or thrombosis.
The endothelium is a major contributor to the inhibitor systems that control platelet
activation.
Eicosanoids
In 1976 Vane’s group reported that the endothelial cells synthesise prostacyclin, a potent
but short-living inhibitor of platelet aggregation and stimulator of platelet disaggregation
(Moncada et al., 1976). In fact, prostacyclin can be considered as a biological opponent of
thromboxane A2 on platelets and the vessel wall, as prostacyclin causes vasodilatation and
inhibition of aggregation, and these actions are antagonized by thromboxane A2 (Bunting,
Moncada and Vane, 1983).
Prostacyclin binds to its specific receptors present on platelets that are linked to the
adenylate cyclase. Stimulation of prostacyclin receptors leads to increased accumulation of
the intracellular cAMP and down-regulation of all pathways involved in amplification of
platelet aggregation (Bunting, Moncada and Vane, 1983). Prostacyclin exerts little influence
on the process of platelet adhesion to subendothelial components of the vessel wall
(Radomski, Palmer and Moncada, 1987a).
Prostacyclin acts as a paracrine inhibitor of platelet activation. It is released close to

the endothelial surface in response to stimulation with various vasoactive mediators
including angiotensins and bradykinin (Nowak et al., 1981). Platelets themselves lack
the capacity to synthesise prostacyclin, however, they may contribute to the endothelial
synthesis of this eicosanoid by generating and releasing the arachidonic acid cyclic
endoperoxides that may be taken up by the endothelial cells for prostacyclin synthesis
(Bunting, Moncada and Vane, 1983).
Some of lipoxygenase metabolites of polyunsaturated fatty acids including 12-
hydroperoxy and 13-hydroxy derivatives of arachidonic and linoleic acids, respectively,
have been shown to inhibit the process of platelet activation (Aharony, Smith and Silver,
1981; Buchanan and Brister, 1991). The precise mode of action of these metabolites on
platelets is not known.
Nitric oxide
Nitric oxide (NO) accounts for the vasodilator activity of endothelium-derived relaxing
factor (EDRF), a non-prostaglandin vasorelaxant substance first described in the endothelial
cells by Furchgott and Zawadzki (1980).
In 1987 we found that cultured endothelial cells released NO to inhibit platelet adhesion,
aggregation and cause dis-aggregation of pre-formed platelet aggregates (Radomski,
Palmer and Moncada, 1987a-d). Nitric oxide is a gaseous mediator synthesised from L-
arginine by the family of isoformic enzymes termed NO synthases (NOS). To date three
isoforms eNOS, iNOS and nNOS have been identified (Radomski, 1995). Although cDNAs
for the respective proteins are found almost in all mammalian cells, under physiological
conditions, eNOS is a major NOS isoform expressed in the endothelial cells (Radomski,
1995). In contrast, inflammation and cell damage are often associated with the expression
of iNOS (Radomski, 1995).
Nitric oxide is generated and released from the endothelial cells both under basal and
agonist-stimulated conditions. Shear stress and pulsatile flow are major stimuli that cause
release of NO under basal conditions (Cooke et al., 1991). These physical forces are also
responsible for the rheological arrangement of platelets in the flowing blood close to the
surface of endothelial cells (see above). It is, therefore, hardly surprising that the endothelial
NO regulates both adhesion and aggregation of platelets in vivo acting locally as a paracrine
mediator (Yao et al., 1992).
In 1990, we also discovered that NO can exert an autocrine influence on platelet
function (Radomski, Palmer and Moncada, 1990). This is due to the intraplatelet NOS
that is regulated by platelet activation generating amounts of NO sufficient for
downregulation of platelet recruitment to the site of the endothelial injury as well as
platelet aggregation (Radomski, Palmer and Moncada, 1990; Bode-Boger et al., 1998;
Freedman et al., 1997).
Ecto-nucleotidase ATP-diphosphohydrolases (ADP-ases)

These enzymes act as biological opponents of ADP, as they metabolise ADP to AMP and
adenosine (Marcus and Safier, 1993). Adenosine can per se inhibit platelet activation
contributing to the overall platelet-inhibitory action of ADP-ases.
Figure 5.3 Platelet haemostasis is regulated as a tightrope balance between the activator and inhibitor mediator
systems.
PHYSIOLOGICAL MODULATION OF PLATELET BEHAVIOUR BY THE

ENDOTHELIUM: THE INTERACTIONS BETWEEN MEDIATORS
Vascular haemostasis is mediated by the interactions between mediators generated in the

endothelium-blood cell microenvironment. Physiological platelet haemostasis is best viewed
as a tightrope balance between mediators. Both interactions between the activator and
inhibitor mediator systems (Figure 5.3) and the interactions within the groups of mediators
(Figure 5.4 and 5) are likely to occur.
Interactions between the Activator and Inhibitor Mediator Systems
System interactions
The best example of the interactions between the activator and inhibitor systems controlling
platelet activation is the generation and release of NO and prostacyclin by platelet-
aggregating agents such as thrombin (Yang et al., 1994). The biological significance of this
generation is to down-regulate the extent of the activator response.
Biological opponents
Interestingly, some of the antagonistic pairs of mediators share the same biosynthetic
pathway. Prostacyclin acts as a biological opponent of thromboxane A2 on aggregation and
vessel wall reactivity and both eicosanoids are synthesised by cyclooxygenase via the
intermediate stage of cyclic endoperoxides. At this stage the pathways separate and
prostacyclin and thromboxane are generated by prostacyclin synthase and thromboxane
synthase, respectively (Hamberg, Svensson and Samuelsson, 1975; Moncada et al., 1976).
This is why inhibition of this enzyme by cyclooxygenase inhibitors such as aspirin and
aspirin-like drugs decrease the levels of both eicosanoids. It has been proposed, however,
Figure 5.4 The activator pathways of platelet aggregation.
Figure 5.5 The inhibitor pathways of platelet aggregation.

that lower doses of aspirin may more selectively inhibit generation of thromboxane A2 in
platelets without affecting generation of prostacyclin in the endothelial cells (Moncada,
1982). The importance of prostacyclin as a biological opponent of thromboxane is further
emphasised by the fact that the antithrombotic activity of thromboxane synthase inhibitors
may be prostacyclin-dependent (De Clerck et al., 1990).
Peroxynitrite is a potent oxidant that is generated from NO and superoxide as a result of
rapid non-enzymatic reaction (Beckman and Tsai, 1994). Peroxynitrite opposes the platelet
inhibitor and vessel wall relaxant effects of NO (Moro et al., 1994; Villa et al., 1994). In
this context it is worth to emphasise that superoxide dismutase that scavenges superoxide
inhibits platelet aggregation (Salvemini et al., 1989).
Synergistic interactions
Synergy of two or more pharmacological agents takes place when their combined effect
exceeds the sum of individual effects. In platelets, a synergistic enhancement of platelet
aggregation has been described during interactions involving ADP and thromboxane (Grant
and Scrutton, 1980). Moreover, subthreshold amounts of NO potentiate the platelet-inhibitory
effects of prostacyclin (Radomski, Palmer and Moncada, 1987d). Inhbition of cGMP-
inhibited cAMP phosphodiesterase by NO has been proposed to account for the synergistic
interactions between these two mediators (Maurice and Haslam, 1990).
REGULATION OF LEUKOCYTE FUNCTION BY ENDOTHELIUM
Leukocytes and Leukocyte Recruitment

Although less numerous than platelets, leukocytes are nucleate blood cells that are several
times larger in size than platelets. The major role of blood leukocyte system is to participate
in various inflammatory and immune reactions including the response to the endothelial
injury. This response of leukocytes is known as leukocyte recruitment.
Leukocyte recruitment
This is the multistep cascade of events that leads to leukocyte presence at the site of
injury. Whereas platelet recruitment occurs both at the arterial and venous vasculature,
leukocyte recruitment is thought to take place mainly in the postcapillary venules (Perry
and Granger, 1991).
The initiating signal for the leukocyte recruitment may be delivered by various factors
including histamine and thrombin, however the subsequent steps of this process appear to
be similar (Kubes, 1995). Blood leukocytes travelling in flowing blood at a relatively high
speed make an initial contact with the endothelial cell lining (tethering), and then move
along the endothelium at greatly reduced velocity by a process described as rolling.
Both leukocyte tethering and rolling are believed to be mediated by up-regulation of the
selectin family of adhesion receptors involving the reactions dependent on P-, L- and E-
selectin (Kubes, 1995). Once leukocytes begin to roll, they can then firmly adhere and
finally emigrate out of the vasculature. The firm adhesion is mediated by leukocyte integrins,
particularly by the CD11/CD18 receptor (Kishimoto and Anderson, 1992). The factors that
Figure 5.6 The leukocyte recruitment in the postcapillary venules.
trigger leukocyte recruitment also cause increased expression of endothelial selectins (Kubes,
1995), and blockade of these receptors by monoclonal antibodies down-regulates the cascade
of leukocyte adhesion to the endothelial lining. Interestingly, some of leukocyte recruitment
appears to be preceded by mast cell activation, histamine release and activation of P-selectin
(Gaboury et al., 1995).
Leukocyte recruitment is often associated with formation of leukocyte-platelet aggregates
(Figure 5.6). These interactions appear to be P-selectin-dependent (McEver, 1991).
Regulation of leukocyte recruitment

The studies using inhibitors of NOS and NO donors have shown that NO may play a crucial
role in regulation of leukocyte recruitment. Indeed, inhibition of NOS with L-NAME
increased leukocyte rolling and adhesion, both effects attenuated by cGMP analogues and
NO donors (Kubes, Suzuki and Granger, 1991). L-NAME-induced increase in leukocyte
adhesion may involve the release of chemoattractants such as platelet activating factor and
leukotriene B4 (Kubes, 1995).
THE INTERACTIONS BETWEEN RED CELLS AND THE

ENDOTHELIUM
Oxygen-carrying function is the main physiological task of red cells that has direct impact
on the endothelial function. Recently, it has been suggested that in addition to oxygen, red
cell haemoglobin may serve as a carrier molecule for NO in the nitrosohaemoglobm complex
(Stamler et al., 1997)
Red Cells and Shear Stress

The importance of red cells for shear stress including changes in platelet function is best
exemplified by the facts that anaemia (characterised by low red cell number and/or
erythrocyte dysfunction) and polycythaemia (characterised by increased number of
erythrocytes) affect haemostasis. Indeed, bleeding is an important characteristic of anaemia
and thrombosis often complicates polycythaemia (Pearson, 1987).
Adhesion of Red Cells to the Endothelial Lining

As discussed before, under physiological conditions red cells occupy central position in
the blood stream and have limited opportunity to interact directly with the endothelial
lining. However, erythrocytes altered by the diseased processes such as sickle cell anaemia
show increased adhesiveness to the endothelial cells (Wick and Eckman, 1996). This
increase is probably mediated via activation of interactions with adhesion proteins
including high molecular weight von Willebrand factor and thrombospondin (Wick and
Eckman, 1996).
CONCLUSIONS
Research over the past 25 years has revolutionised the views on the role of endothelial cells
in regulation of vessel wall reactivity and haemostasis. It is now clear that the endothelial
cells are the “maestro” of the vasculature and play a key role in regulation of vascular
haemostasis.
ACKNOWLEDGEMENTS
This work was supported by a grant from Medical Research Council of Canada. MWR is a
Scholar of the Heritage Foundation for Medical Research.
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Yang, Z., Arnet, U., Bauer, E., von Segesser, L., Siebenmann, R., Turina, M. and Luscher, T.F. (1994) Thrombin-
induced endothelium-dependent inhibition and direct activation of platelet-vessel wall interaction. Role of
prostacyclin, nitric oxide, and thromboxane A2. Circulation, 89, 2266–2272.
Cardiovascular disease still accounts for the majority of morbidity and mortality in Western countries. Most
forms of cardiovascular disease involve atherosclerotic vascular changes in the coronary, cerebral, renal and
peripheral circulation leading to angina pectoris and myocardial infarction, stroke, renal failure and claudicatio.
The endothelium is a monolayer of cells lying on the vascular wall, which for years was considered to be only
a protective barrier. In the past two decades, however, it has been shown that the endothelium plays indeed an
active role in the regulation of vascular smooth muscle cell function and tone. The endothelium is in a strategic
anatomical position within the blood vessel wall, located between the circulating blood and vascular smooth
muscles. It can respond to mechanical and hormonal signal from the blood. Of particular importance is the fact,
that the endothelium is a source of mediators which can, in a predominantly paracrine fashion, modulate the
contractile state and proliferative responses o vascular smooth muscle cells, platelet function, coagulation as well
as monocyte adhesion. The important role taken by the endothelium in the control of vascular tone is do to its
capacity to release both vasodilating and vasoconstricting substances (Lüscher, 1990; Yanagisawa et al., 1988;
Furchtgott, 1980).
The endothelium plays a protective role as it prevents adhesion of circulating blood cells, keeps the vasculature
in a vasodilaied state and inhibits vascular smooth muscle proliferation and migration. In disease states, on the
other hand, endothelial dysfunction contributes to enhanced vasoconstrictor responses, adhesion of platelets and
monocytes as well as proliferation and migration of vascular smooth muscle cells, all as events known to occur in
atherosclerosis.
Endothelial function is impaired in certain cardiovascular conditions including atherosclerosis and in the
presence of risk factors such as including atherosclerosis (Zeiher et al., 1993), diabetes (Johnstone, 1993), smoking
(Zeiher et al., 1995), hypercholesterolemia (Chowienczyk et al., 1992, Drexler et al., 1991, Creager et al., 1992),
aging (Taddei et al., 1995), menopause (Taddei et al., 1996a) and hypertension (Taddei et al., 1993, Taddei et al.,
1997c, Vallance et al., 1992a).
Hypertension is an important risk factor for the development of cardiovascular disease (MacMahon et al.,
1990) (Figure 6.1). It is associated with an increase in pressure on the arterial side of the circulation, most due
to elevated peripheral resistance, determined by the contractile state of the resistance arteries with a diameter
of 200 µm or less. The resistance arteries are influenced by neuronal stimulation (in particular from the
sympathetic nervous system), by circulating hormones and paracrine and autocrine mechanisms with the blood
vessel wall.
Normally the vessel wall is in a constant state of vasodilation due to the basal formation of nitric oxide (NO)
by endothelial cells (Rees et al., 1989). This dominant vasorelaxing propriety of the endothelium may act as a
compensatory mechanism in an attempt to limit vascular resistance. On the other hand, the vascular endothelium
might be involved directly to increase peripheral resistance, via an enhanced release of constricting factors and/
or a decreased release of relaxing factors. Furthermore, the endothelium may importantly contribute to the vascular
complications of hypertension, as it becomes dysfunctional (Blot et al., 1994; Palmer et al., 1992).
6 Endothelial Dysfunction and Hypertension
Roberto Corti, Isabella Sudano, Christian Binggeli, Eduardo Nava,

Georg Noll and Thomas F.Lüscher
Division of Cardiology, University Hospital Zürich, CH-8091 Zurich, Switzerland
THE PHYSIOLOGIC FUNCTION OF THE ENDOTHELIUM
The endothelium is an organ with a very active secreting activity (Figure 6.1). Endothelial
cells release numerous vasoactive substances (i.e. nitic oxide; NO, endothelin; ET,
prostacyclin and endothelium-derived hyperpolarising factor; EDHF) which regulate vascular
smooth muscle and trafficking blood cells. In response to many stimuli, such as increased
shear forces exerted by increased blood flow (Lüscher, 1990; Yanagisawa et al., 1988;
Furchtgott, 1980) the endothelium releases NO, which is a potent vasodilator which also
inhibits cellular growth and migration. In addition, NO possesses antiatherogenic and
thromboresistant proprieties by preventing platelet aggregation and adhesion.
NO is formed from L-arginine by oxidation of its guanidine-nitrogen terminal (Palmer
et al., 1988; Moncada, 1992). The catalyzing enzyme NO-synthase (NOS) is constitutively
expressed and exists in several isoforms in endothelial cells, platelets, macrophages, vascular
smooth muscle cells, and the brain. NO is rapidly inactivated by free radicals, so that its
half-life is only of few seconds.
Endothelial cells also release the vasoconstrictor ET. ET exists in three isoforms:
ET-1, ET-2 and ET-3. Endothelial cells produce ET-1 exclusively (Yanagisawa et
al., 1988).
Translation of messenger RNA (mRNA) generates pre-pro-endothelin, which is
covered to big ET; its conversion to the nature peptide ET-1 by the ET-converting enzyme
(ECE) is necessary for the development of full vascular activity. The circulating levels
of ET-1 are very low; this suggests that little of the peptide is formed physiologically,
which may be due to the absence of stimuli for production of ET, the presence of potent
inhibitory mechanisms, or its preferential release abluminally toward smooth muscle
cells (Wagner et al., 1992).
Due to degradation by endopeptidases in the plasma, lung, and kidney circulating ET-1
has a short half-time, of about 4 to 7 minutes (de Nucci et al., 1988).
Endothelin can stimulate the release and action of NO via a distinct endothelial receptor
(ETB-receptor). This explains why ET causes a transient vasodilation at lower concentrations,
which precedes its pressor effect (Yanagisawa et al., 1988; Wright and Fozard, 1988; Kiowski
et al., 1991).
Moreover in pathological conditions, the endothelium can also produce other
endothelium-derived contracting factors (EDCFs), which are mainly cyclooxygenase-dependent
Correspondence: Thomas F.Lüscher, M.D., F.E.S.C., Professor and Head of Cardiology, University Hospital,
CH-8091 Zurich, Switzerland. Tel: 0041 1 255 22 16; Fax: 0041 1 255 44 01.
109
110 R.Corti et al.
Figure 6.1 Endothelium-derived vasoactive substances: Various blood- and platelet-derived substances can activate
specific receptors (open circles) on the endothelial membrane to release relaxing factors such as nitric oxide
(NO), prostacyclin (PGI2) and a hyperpolarizing factor (EDHF). Furthermore contracting factors are released
such as ET (ET1), angiotensin (A), and thromboxane AII (TXAII) as well as prostaglandin H2 (PGH2).
=superoxide (from Lüscher, Noll, Braunwald’s Heart Disease 1997).
prostanoids (i.e. thromboxane A2: TXA2, and prostaglandine H2: PGH2) or superoxide
anions.
EXPERIMENTAL MODELS OF HYPERTENSION
Inhibitors of NO production cause endothelium-dependent contractions of isolated arteries

(Tschudi et al., 1994b), decrease blood flow (Vallance et al., 1989; Joannides et al., 1995a)
and induce pronounced and sustained hypertension when infused intravenously or given
orally in vivo (Rees et al., 1989; Moreau et al., 1995).
Alteration in endothelium-dependent relaxation in hypertension is not uniform and
depends on the model of hypertension as well as the vascular bed studied (Figure 6.2). In
some vascular beds of hypertensive rats such as the aorta, mesenteric, carotid and cerebral
vessels, endothelium-dependent relaxation is impaired (Luscher, 1994a; Luscher and
Vanhoutte, 1986a; Dohi et al., 1990; Diederich et al., 1990; Dohi et al., 1991; Luscher,
1992; Luscher, 1994b; Kung and Luscher, 1995; Lüscher, 1986). In contrast, in coronary
and renal arteries of spontaneously hypertensive rats, endothelial function does not seem to
be affected by high blood pressure (Tschudi et al., 1991; Luscher, 1991). On the other
hand, depending on the animal model and the vascular bed endothelium-dependent
contractions have been documented. Since cyclooxygenase inhibitors and thromboxane
receptor antagonists can inhibit this response, the most likely contractile factors are
thomboxane A2 and/or prostaglandin H2 (Kung and Luscher, 1995; Noll et al., 1997a).
Acute, pharmacologically-induced elevations in blood pressure cause an increased
release of NO and, on the other hand, drops in pressure are followed by a decreased
Endothelial Dysfunction and Hypertension 111
Figure 6.2 Endothelial dysfunction in experimental hypertension: While in the SHR (left panel) NOS is
upregulated and NO is inactivated by , in salt-related hypertension (Dahl rats) NO is produced in lesser
amounts, while the ET system is up-regulated. Abbreviations as Figure 2 (from Lüscher, Noll, Braunwald’s
Heart Disease 1997).
production, suggesting that high blood pressure up-regulates NO production and vice
versa (Nava, 1994). The mechanism by which high blood pressure leads to an increased
production of NO is not clear yet. It is known that the release of NO by endothelial cells
can be altered by changes in blood flow (Joannides et al., 1995a, Buga et al., 1991) and
that mRNA and protein for cNOS can be induced by mechanical forces (Sessa et al.,
1994). It is likely that not only shear stress, but also other mechanical factors such as
blood pressure itself and pulsatile stretch (Hishikawa and Luscher, 1997) contribute to
this phenomenon.
The activity of cNOS is higher in mesenteric resistance arteries obtained from
spontaneous hypertensive rats (SHRs) compared to age-matched normotensive rats (Noll
et al., 1997b). Moreover, the concentration of the oxidative product of NO, nitrate,
measured by HPLC and capillary electrophoresis, is higher in the hypertensive rats as
compared to their normotensive controls (Noll et al., 1997b). In contrast, prehypertensive
young SHRs exhibit similar nitrate levels as age-matched normotensive controls. These
observations demonstrate that the basal release of NO is increased in rats with spontaneous
hypertension and that this increased production is directly related to the increased blood
pressure of the animals.
Further studies demonstrated that the level of cyclic GMP in mesenteric resistance
arteries is similar in SHR and in Wystar-Kyoto rats (WKY) (Noll et al., 1997b). Moreover,
the NO-dependent vasodilator tone, assessed by the blood pressure effects of L-
nitroarginine methylester (L-NAME), is not higher in hypertensive rats as would be
expected in a situation in which the production of NO is increased. The capacity of vascular
smooth muscle cells of hypertensive rats to respond to NO, on the other hand, must be
fully maintained as organic nitrates lower blood pressure in a similar fashion in both
112 R.Corti et al.
strains of rats and relaxations to sodium nitroprusside are enhanced in this condition
(Diederich et al., 1990). These studies indicate that the endogenously-produced NO is
increased in spontaneous hypertension, but is not able to properly raise cyclic GMP levels
in the vascular smooth muscle cells of these animals.
Hence, it appears that in spontaneous hypertension, an additional unknown event takes
place that blunts the hemodynamic actions of NO. The hypertrophied and fibrotic intimal
layer of hypertensive vessels may represent a physical barrier for NO. The chemical
environment that NO has when released can also determine its fate. In this line, oxidative
stress has been proposed to play a role in the pathogenesis of some cardiovascular diseases
including hypertension (Ohara et al., 1993, Nabel et al., 1988, Nakazono et al., 1991).
Recent experimental evidence using a porphyrinic microsensor for direct measurement of
NO has demonstrated that in the presence of superoxide dismutase, NO release from isolated
resistance vessels is improved in the stroke-prone SHR (Tschudi et al., 1996). Thus, a higher
production of oxidative radicals like superoxide anion by a dysfunctional NO-synthase
(Cosentino et al., 1998) or a diminished activity of superoxide dismutase may account for
an increased degradation of NO.
NO production might be heterogeneously affected in different forms of hypertension
(Lüscher, 1988). Indeed, in Dahl salt-sensitive rats endothelium-dependent relaxations are
impaired, but not those to sodium nitroprusside (Lüscher, 1988; Luscher et al., 1987a). In
contrast to spontaneous hypertension, in salt-sensitive hypertension no release of
vasoconstrictor prostanoids can be demonstrated (Luscher et al., 1987a). This suggests that
a decreased NO production could contribute to the pathogenesis of this form of hypertension.
Similar experimental findings suggest that ET may be differently involved in different
forms of hypertension. In fact, in some animal models of hypertension, such as the DOC A
salt hypertensive rat, ET receptor blockade causes marked reductions in blood pressure,
which is also associated with regression of vascular hypertrophy (Li et al., 1994, Schiffrin
et al., 1995). In keeping with that, ET-1 secretion is augmented in cultured endothelial cells
from DOCA-salt hypertensive rats (Takada et al., 1996). Accordingly, ET antagonists lower
blood pressure in salt-depleted monkeys (Clozel et al., 1993).
However, the effects of ET-antagonism in other experimental models of hypertension,
notably SHRs, are less clear. In the SHR, both circulating and vascular ET as well as ET
tissue content of the renal medulla are reduced (Li and Schiffrin, 1995a; Kitamura et al.,
1989). In contrast, in the stroke-prone SHR the ET axis is activated and ET antagonism
significantly reduces blood pressure and prevents cardiac and vascular hypertrophy (Stasch
et al., 1995; Chillon et al., 1996). As in Dahl salt-sensitive rats, ET levels are increased and
ET-antagonists lower blood pressure, this indicates that the ET system is particularly activated
in severe, salt sensitive (low-renin) hypertension (Barton et al., 1998).
In one-kidney, one-clip (low-renin) hypertension (Li et al., 1996) and two-kidney, two-
clip acute renal failure (Ruschitzka et al., 1998) the circulating and tissue ET-1 system is
activated. At variance, the ET expression is augmented only in the late phase of two-kidney,
one-clip Goldblatt (high-renin) hypertension resembling true renovascular hypertension
and activation of the renin-angiotensin-system in man (Sventek, 1996). In contrast, two
weeks administration of angiotensin II increases the production of ET in the blood vessel
wall of the rat (Moreau et al., 1991 a; d’Uscio et al., 1997). Most interestingly, selective
ETA receptor antagonism reduces blood pressure and in particular vascular hypertrophy
(Moreau et al., 1997a) and endothelial dysfunction (d’Uscio et al., 1997) under these
experimental conditions. These data strongly suggest that ET antagonists may be of particular
value in conditions of increased activity of the renin-angiotensin system. This is consistent

with additional hypotensive effects of ET antagonists in hypertensive dogs already treated
with an ACE inhibitor (Donckier et al., 1997). However, the discrepancy between the
beneficial effects of selective ETA receptor blockade in angiotensin II induced hypertension
and the lack of effects in two-kidney, one-clip model is difficult to reconcile, but may be
due to a lesser extent of activation of the tissue (rather than circulating) renin-angiotensin
and ET system.
In NO-deficient hypertension induced by L-NMMA or L-NAME, ET production is
enhanced, but the peptide is only involved in the early, but not chronic phase of
hypertension (Sventek et al., 1997; Moreau et al., 1997b). Recently, a role of ET was
also suggested in fructose-fed hypertensive rats exhibiting hyperinsulinemia and insulin
resistance, as chronic combined ET blockade reduces blood pressure in this experimental
model of hypertension (Verma et al., 1995). Interestingly, hepatic overexpression of
preproET-1 in rats also resulted in elevation of blood pressure that was reduced by an
ETA antagonist (Niranjan, 1996).
To further elucidate the role of ET, transgenic and gene knock-out rats have been
developed. Endothelin-2 transgenic rats exhibit elevated ET plasma levels, but do not develop
hypertension (possibly because of the activation of compensatory vasodilator mechanisms)
(Hocher et al., 1996). Surprisingly, ET-1 gene knock-out mice are actually hypertensive
(Kurihara, 1993b). It is likely that the small increase in blood pressure in ET knock-out
mice is related to hypoxia and in turn activation of the sympathetic nervous system in these
animals. The finding that ET knock-out rats have profound malformations of the throat
indicates that the peptide may be importantly involved in the development of these organs
(Kurihara, 1993a).
Other vasoactive mediators are also candidates to contribute to endothelial dysfunction
in hypertension. Indeed, the responses to angiotensin I and II are increased in spontaneously
hypertensive rats (Tschudi and Luscher, 1995), and in addition platelets and platelet-derived
substances (ADP, ATP, serotonin), known to stimulate the formation of EDCFs (Luscher
and Vanhoutte, 1986b), may lead to increased peripheral vascular resistance and also to
complications of hypertension.
HYPERTENSION IN MAN
Role of Nitric Oxide

Experiments in humans have demonstrated a diminished basal and stimulated NO
production (Taddei et al., 1993; Kiowski et al., 1991; Calver et al., 1992; Panza et al.,
1990). The decrease in forearm blood flow induced by L-NMMA (reflecting basal NO
formation) is smaller in hypertensive than in normotensive patients (Calver et al., 1992).
L-NMMA infusion however provides only indirect evidence and no analytical
determinations of basal NO production. Benjamin and co-workers demonstrated that
indeed plasma levels of NO are reduced in patients with essential hypertension (Forte et
al., 1997) (Figure 6.3). The stimulated release of NO as assessed by the vasodilator effects
of acetylcholine in the forearm circulation of patients with essential, renovascular or
endocrine hypertension are reduced in all but one study (Forte et al., 1997; Panza et al.,
114 R.Corti et al.
Figure 6.3 Plasma nitric oxide levels in normotensive controls and patients with essential hypertension (from
Forte et al., Lancet, 1997).
1990; Panza et al., 1993). The reasons for the negative results in the study by Cockcroft
et al. is very likely due to the relatively low dosages of acetylcholine infused and/or
heterogeneity in endothelial dysfunction in different patients. Similar findings have been
obtained in the coronary circulation, particularly in the presence of left ventricular
hypertrophy (Zeiher et al., 1993; Cockcroft et al., 1994).
In patients with essential hypertension, the impaired response to acetylcholine in the
forearm circulation can be improved by indomethacin, suggesting that cyclooxygenase-
dependent vasoconstrictor prostanoids also contribute to impaired endothelium-dependent
relaxation in hypertensive patients (Taddei et al., 1993). Moreover, besides endothelium-
derived contracting factors (EDCFs) such as TXA2 and PGH2, oxygen-free radicals can
play an important role in endothelial dysfunction in hypertension (Tschudi et al., 1996;
Taddei et al., 1998a).
Interestingly, there appears to be a difference in endothelial dysfunction between primary
and secondary forms of hypertension. In essential hypertension a normalization of blood
pressure values do not improve the endothelial function, in contrast to what append with
secondary forms of hypertension (Taddei et al., 1993). Moreover, in essential hypertension
no correlation between blood pressure values and degree of endothelial dysfunction was
found (Taddei et al., 1993).
NO plays an important role in renal function. Indeed, the kidney is extremely sensitive
to NO inhibition as very low doses of L-arginine analogues, which do not affect blood
pressure, diminish diuresis, natriuresis and renal plasma flow (Lahera et al., 1991; Salazar
et al., 1992). It is possible that in some forms of hypertension, minimal alterations in the
renal production of NO, which do not alter endothelium-dependent relaxations, lead to
systemic hypertension due to a change in the management of body fluids by the kidney.
Moreover, it has been recently shown that renal failure is also associated with an accumulation
of an endogenous inhibitor of NO synthesis, asymmetrical dimethylarginine (Vallance et
al., 1992b), which could also explain the increase in peripheral resistance and hypertension
observed in these patients.
Role of Endothelin
Whether or not ET is involved in hypertension is still controversial (Luscher et al., 1993).

Because of its vasoconstrictor action and its effects on vascular hypertrophy, ET-1 has also
been implicated in the pathogenesis and/or the maintenance of hypertension. However,
whether ET production is altered in human hypertension remains still elusive (Luscher et
al., 1993; Vanhoutte, 1993). Although few studies found increased plasma levels of ET in
hypertensives, many others found no differences as compared to controls. Interestingly,
patients with ET-secreting hemangioendotheliomas have huge increases in plasma ET and
are hypertensive (Yokokawa et al., 1991). Plasma ET concentrations are also elevated in
women with preeclamsia (Kamoi et al., 1990; Schiff et al., 1992; Nova et al., 1991). Increased
ET levels in African-Americans who often present with severe and salt-sensitive (low-renin)
hypertension, point to the fact that severity of the blood pressure increase as well as salt-
sensitivity (as suggested by the experimental models; see above) are important denominators
for the activation of the ET system in hypertension (Ergul et al., 1996). However, circulating
ET may not reflect local levels of the peptide, as in the blood vessel wall ET is primarily
released abluminally (Wagner et al., 1992). Recent studies, using inhibitors of the ET
converting enzyme or ET receptor antagonists, suggest that ET does contribute to blood
pressure elevation in certain forms of hypertension in laboratory animals and in humans
(McMahon et al., 1991; Nishikibe et al., 1993; Haynes et al., 1996).
Infusion of exogenous ET does increase blood pressure in experimental animals and in
man (Pernow et al., 1996). In healthy subjects, ET-1 receptor antagonism as well as infusion
of the inhibitor of ET converting enzyme phosphoramidone increases forearm blood flow
and lowers blood pressure indicating a role of ET-1 in the regulation of vascular tone
(Kiowski et al., 1991; Haynes and Webb, 1994; Kaasjager et al., 1997). In essential
hypertensives, ET-1-induces an increase in blood pressure and systemic vascular resistance,
while cardiac index and natriuresis is reduced (Kaasjager et al., 1997). Moreover, the
vasoconstrictor response to ET is increased in the human hand vein circulation of patients
with essential hypertension (Haynes et al., 1995). Interestingly, normotensive offsprings of
hypertensive parents exhibit enhanced plasma ET responses to mental stress indicating that
genetically determined activation of the ET system is already present at this early stage of
disease (Noll et al., 1996). Furthermore, ET-1 gene expression is enhanced in small arteries
of patients with moderate to severe hypertensive, whereas expression is similar in control
subjects and untreated mild hypertensives (Schiffrin et al., 1997). In line with these studies
chronic therapy with the ETA-/ETB-receptor antagonist bosentan does indeed lower blood
pressure in patients with essential hypertension to quite a similar degree as the ACE-inhibitor
enalapril (Krum et al., 1998) (Figure 6.4) (see below).
116 R.Corti et al.
Figure 6.4 Effects of the ETA-/ETB-receptor antagonist bosentan in patients with essential hypertension as compared
to the ACE-inhibitor enalapril (from Krum H et al. N. Engl. J. Med. 1998).
ANTIHYPERTENSIVE THERAPY AND ENDOTHELIAL FUNCTION
Endothelial dysfunction is a common feature of several pathological processes including

hypertension, hyperlipidemia and atherosclerosis. Drugs that could improve the endothelial
function or enhance alternative pathways to substitute for the alterations of the release of
endothelial mediators may have a potential advantage in the treatment of these pathological
conditions. Several antihypertensive agents can prevent and reverse impaired endothelium-
dependent relaxations in large conduit arteries (Luscher et al., 1987b; Tschudi et al., 1994a)
as well as in resistance arteries of hypertensive rats (Dohi et al., 1994). Since diuretics,
calcium antagonists, ACE-inhibitors and angiotensin (All) receptor antagonists improve or
normalize the endothelial dysfunction in hypertensive rats, the blood pressure lowering
properties of these agents appear to be involved in this effect. However, additional pressure-
independent effects of the various drugs cannot be ruled out and are described for each
class of pharmacological agents.
Nitrates
Nitrovasodilators such as nitroglycerin, sodium nitroprusside and linsidomine exert their
vasodilator effects by releasing NO from their molecule (Feelisch, 1987), therefore acting
through a mechanism identical with that of endogenously produced NO. Hence, these drugs
may be particularly useful at sites of reduced vascular formation of NO such as diseased
coronary arteries. Of particular interest is the fact that the endogenous production of NO
reduces the sensitivity of the blood vessel wall to nitrates and nitrovasodilators (Pohl, 1987).
Conversely, in human arteries devoid of endothelium, the concentration-relaxation curve
to linsidomine is shifted to the left (Luscher et al., 1989; Joannides et al., 1995b). This
could ensure a more selective action of these drugs to dysfunctional vascular beds.
In addition to be more effective in sites of reduced NO formation, the nitrovasodilators
seem to be more effective in larger epicardial coronary arteries rather than in smaller coronary
arteries (Sellke et al., 1990). This may be due to the fact that small coronary arteries are
unable to transform nitroglycerin into active molecules. Since adenosine, which is primarily
relaxing small vessels, may precipitate ischemia, this selective efficacy of nitrovasodilators
may have important clinical implications. A clinically important drawback, however, is the
fact that most nitrovasodilators are prone to tolerance.
Calcium Antagonists
Under acute conditions, calcium antagonists do not affect the release of endothelium-derived
vasoactive substances, although the production of these factors in endothelial cell is
associated with an increase in intracellular calcium (Vanhoutte, 1988). Indeed, endothelial
cells do not appear to possess voltage-operated calcium channels. During their chronic
administration, however, these antihypertensive agents improve endothelial function in
experimental animals as well as in patients with essential hypertentsion (Tschudi et al.,
1994a; Takase et al., 1996; Kung et al., 1995a; Taddei et al., 1997b; Taddei et al., 1997a).
Since this beneficial effect is also observed in L-NAME-induced hypertension, in which
the synthesis of NO is inhibited, alternative pathways of endothelium-dependent relaxation
(i.e. enhanced release of hyperpolarizing factor; EDHF) may be involved (Takase et al.,
1996; Kung et al., 1995a). Calcium antagonists may also facilitate the effects of endothelium-
derived relaxing factors at the level of vascular smooth muscle, as suggested by an enhanced
sodium nitroprusside-induced relaxation under certain conditions (Kung et al., 1995b).
Moreover, some calcium-antagonists (verapamil, nifedipine, amlodipine, isradipine and
lacidipine) show antioxidant activity in vitro which may be relevant to improve endothelial
function in the presence of increased oxidative stress. In contrast, for diltiazem no antioxidant
effect could be found (Lupo et al., 1994).
In addition, calcium antagonists seem to interfere with the vasoconstrictor effects of ET
and cyclooxygenase-derived contracting factors (Noll et al., 1991). Indeed, in the porcine
coronary artery ET receptors are linked to voltage-operated calcium channels via a G-protein
and calcium-antagonists attenuate ET-induced vasoconstriction in this blood vessel (Goto
et al., 1989). Furthermore, in the human forearm circulation, ET-1 induces potent contraction,
which is prevented by nifedipine and verapamil (Kiowski et al., 1991). However, there
may be regional differences, as calcium antagonists are ineffective in inhibiting ET-induced
contraction in some vessels, like the internal mammary artery (Yang et al., 1990). It is not
clear, however, if the usual clinical doses of calcium antagonists are sufficient to antagonize
endogenous ET-induced contraction.
Angiotensin-converting Enzyme Inhibitors

The angiotensin-converting enzyme (ACE) is mainly located on the endothelial cell
membrane where it transforms angiotensin I into angiotensin II and breaks down bradykinin,
a potent stimulator of the L-arginine and cyclooxygenase pathways (Palmer et al., 1987).
Therefore, ACE inhibitors not only prevent the formation of a potent vasoconstrictor with
proliferative properties, but also increases the local concentration of bradykinin and, in
turn the production of NO and prostacyclin (Wiemer et al., 1991). This latter effect may
participate in the protective effects of the ACE inhibitors by improving local blood flow
and preventing platelet activation. Accordingly, pretreatment of human saphenous vein and
coronary artery with an ACE inhibitor enhances endothelium-dependent relaxation to
118 R.Corti et al.
bradykinin (Yang et al., 1993; Auch Schwelk et al., 1992). The decreased degradation of
bradykinin could therefore explain the improved endothelial function observed with ACE
inhibitors in normotensive and particularly in hypertensive rats (Dohi et al., 1994; Kahonen
et al., 1995; Bossaller et al., 1992). However, the improvement of the endothelial function
by ACE-inhibitors in L-NAME-induced hypertension suggest that they may also enhance
other endothelium-dependent mechanisms (i.e. enhanced release of EDHF), since the activity
of the enzyme NOS is inhibited in this experimental model (Kung et al., 1995a; Takase et
al., 1996). The mechanism used by ACE-inhibitors also seem to require some time to develop
and cannot be reproduce in acute conditions, again suggesting that another mechanism
than ACE inhibition, which is rapid, is involved (Takase et al., 1996).
In contrast to the striking improvements obtained in experimental models of
hypertension, data of studies in hypertensive patients are still controversial. ACE-inhibitors
seem to improve endothelial function in subcutaneous arteries (Schifrrin and Deng, 1995),
epicardial arteries [Mancini, 1996 #134] and renal circulation (Mimran et al., 1995). In
the forearm circulation, on the other hand, treatment with captopril and enalapril (Creager
and Roddy, 1994) or cilazapril (Kiowski et al., 1996) failed to improve vasodilation to a
muscarinic agonist, while lisinopril selectively improves the vasodilating response to
bradykinin without restoring NO bioavailability (Taddei et al., 1998b). The reasons for
this discrepancy between results obtained in experimental models of hypertension and
studies in hypertensive patients are not clear at the present time. This discrepancy may
originate from the fact that endothelial dysfunction may be treated at a much later stage
in patients than in the rat.
Alternatively, prolonged therapy may be required to restore the endothelial function in
hypertensive patients. Interestingly, in patients with coronary artery disease 6 months
treatment with the ACE-inhibitor quinapril improved endothelial function of epicardial
coronary arteries (Mancini et al., 1996).
Angiotensin II Receptor Antagonists

The recently developed All-receptor antagonists (AT1-receptor blockers) may have
advantages, as they are more potent inhibitors of the angiotensin II vasoconstrictor axis
than ACE-inhibitors (Timmermans et al., 1993). These new drugs are also not associated
with cough, a side effect of ACE-inhibitors generally attributed to the diminished breakdown
of bradykinin. However, if indeed the concomitant stimulation of the L-arginine NO pathway
by bradykinin also proves to be an important property of ACE-inhibitors, All-receptor
antagonists would lack this beneficial effect.
In the experimental model of angiotensin II-induced hypertension losartan enhanced
endothelial-dependent relaxation to acetylcholine and prevents the increase in tissue ET-1
content, suggesting that AT1 receptor blocker can modulate tissue ET-1 in vivo (d’Uscio et
al., 1998). In addition, losartan also blocks the angiotensin-induced production of oxygen-
derived free radicals in this model (Rajagopalan et al., 1996).
In aortic rings obtained from SHR, prolonged antihypertensive treatment with losartan
reverses endothelial dysfunction not only by enhancing NO-dependent relaxation but also
by reducing formation of cycloaxygenase-dependent EDCFs (Rodrigo et al., 1997). Similar
results were also found with captopril as an antihypertensive agent (Rodrigo et al., 1997).
The effects of All antagonists on endothelial dysfunction in human hypertension are
still unknown. As expected in normotensive and hypertensive men the intrabrachial
Table 6.1 Endothelin Receptor Antagonists
infusion of an AT1-receptor blocker (losartan) inhibits the angiotensin H-induced

vasoconstriction (Baan et al., 1998).
Endothelin Antagonists
In recent years a large number of ET-receptor antagonists have been developed (Table 6.1).
In many experimental models of hypertension, these molecules are not effective to lower
blood pressure (Li and Schiffrin, 1995b). As discussed above, they have a modest
antihypertensive efficacy in DOCA-salt hypertensive rats, although they have a more
profound effect on to prevent vascular hypertrophy (Li et al., 1994). These antagonists also
are able to prevent L-NAME-induced hypertension acutely and early on in the chronic
phase of blood pressure elevation but eventually a similar increase in blood pressure is
noted in spite of ETA-/ETB-blockade70. This is surprising if one considers the negative
feedback exerted by NO on ET-1 release that is blocked in this model (Richard et al., 1995;
Donckier et al., 1995).
As studies in humans demonstrated that ETA- as well as combined ETA-/ETB-receptor
blockade increases blood flow in the forearm (Haynes and Webb, 1994) as well as in the
human skin microcirculation ET must contribute to the regulation of the cardiovascular
system also in man (Wenzel et al., 1994). Indeed, intravenous infusion of the ETA-/ETB-
receptor antagonist TAK-044 in humans with preserved left ventricular function lowers
peripheral vascular resistance, blood pressure and increases cardiac output and heart rate
(Sutsch, 1997). Similarly, intravenous infusion of the ETA/B-receptor antagonist bosentan
in patients with coronary artery disease lowers blood pressure under acute conditions
120 R.Corti et al.
(Wenzel, 1998). Bosentan does exhibit a pronounced antihypertensive activity in patients

with essential hypertension similar to that exerted by the ACE-inhibitor enalapril (Figure
6.5) (Krum et al., 1998). This strongly suggests that indeed ET is involved in human
hypertension and may provide a new therapeutic approach to treat this condition. Of
particular interest obviously in this context is the fact that ET antagonist are particularly
efficacious in reversing vascular functional and structural changes in experimental
hypertension.
The type of antagonist (ETA-receptor selective or combined ETA/B-receptor antagonists)
that would be more effective is still a matter of debate. Most of the first generation molecules
were specific ETA-receptor antagonists (Ihara et al., 1991; Itoh et al., 1993; Sogabe et al.,
1993). Combined ETA/B-receptor antagonists are now also available (Clozel et al., 1993;
Clozel et al., 1994; Luscher, 1993; Ikeda et al., 1994; Ohlstein et al., 1994) and they may
prove to be better therapeutic agents, since in several blood vessels, including humans vessels,
both ETA- and ETB-receptors mediate vasoconstriction (Sumner, 1992; Seo et al., 1994;
Ihara et al., 1991). However, these non-selective antagonists also block the endothelium-
dependent vasodilatation of ET, by blocking ETB-receptors (Clozel et al., 1994; Takase et
al., 1995). Indeed, in L-NAME hypertension selective ETA-blockers (i.e. LU 135252)
improve endothelial dysfunction (in spite of no antihypertensive efficacy) (D’uscio, 1998),
while the ETA/B-receptor antagonist bosentan is ineffective (Moreau et al., 1997b). Endothelin
converting enzyme inhibitors may also prove to be valuable therapeutic agents, although
their development lags behind the receptor antagonists.
CONCLUSIONS
Endothelial dysfunction does occur in experimental and human hypertension and may be
relevant both for development of high blood pressure as well as for its cardiovascular
complications (Figure 6.5). Endothelial dysfunction involves a decreased basal and
stimulated release of nitric oxide, enhanced release of endothelium-derived contracting
factors such as thromboxane A2/prostaglandin H2 and most likely also ET-1. Whether or
not it is a primary or secondary phenomenon as suggested by experimental models and
studies in patients with secondary hypertension remains uncertain. Indeed, in essential
hypertensive patients endothelial dysfunction may represent at least in part a primary
phenomenon as it already occurs in normotensive offspring of hypertensive (Luscher, 1994a;
Dohi et al., 1990; Lockette et al., 1986; Taddei et al., 1996b).
Endothelial dysfunction in hypertensive patients may be particularly important it is further
aggravated by other risk factors such as hypercholesteremia, smoking and diabetes, very
much along with the cardiovascular complications.
Blood pressure lowering does normalize endothelial dysfunction in experimental
models of hypertension, while in patients with essential hypertension it is may be more
difficult to achieve. Studies with a larger number of patients and prolonged treatment
periods are required to determine to what extent there are differences between different
antihypertensive drugs and their capacity to reverse endothelial dysfunction. Furthermore,
studies are required to link endothelial dysfunction with the clinical events occurring
later in the disease process.
Figure 6.5 Relation of high blood pressure with cardiovascular complications.
ACKNOWLEDGEMENTS
Swiss National Foundation (TF Lüscher Nr. 32.52069.97), G Noll (Nr. 32.52690.97) and
the Swiss Heart Foundation.
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Patients with chronic heart failure are hemodynamically characterized by increased vasoconstriction and a reduced
vasodilator response to exercise. In addition to various compensatory neurohumoral mechanisms there is evidence
that the endothelium plays an important role in the abnormal vasodilator response. This evidence comes from
studies investigating the microvascular response to regional, intrarterial administration of the endothelium-dependent
vasodilator acetylcholine which found that the vasodilator response and, therefore, bioavailability of nitric oxide
was impaired in the microcirculation of the leg, forearm, and myocardium of patients with chronic heart failure.
The mechanisms underlying this abnormal response are not entirely clear but may reflect a muscarinic receptor
abnormality. Since conduit artery vasodilation during hyperemic blood flow is also impaired and since this response
is not dependent on muscarinic receptor activation this possibility appears to be unlikely. However, impaired
smooth muscle responsiveness to nitric oxide stimulation, impaired L-arginine availability or utilization, endothelial
release of vasoconstricting prostanoids, increased nitric oxide degradation and reduced nitric oxide synthase activity
have all been implicated in this impaired response. In addition, the vasoconstrictor activity of endothelin-1 seems
to play an important role in the regulation of tone in chronic heart failure although the importance of different
endothelin-receptors is not clear yet.
Key words: Heart failure, endothelium, acetylcholine, nitric oxide, L-arginine, substance P, endothelin-1
7 Endothelial Control of Vascular Tone in Chronic Heart
Failure
Wolfgang Kiowski1 and Helmut Drexler2

1
Divisions of Cardiology, University Hospital Zurich, CH-8091 Zurich, Switzerland
2
Medizinische Hochschule Hannover, Germany
INTRODUCTION
It is increasingly recognized that the endothelium plays an important role in the control of
vascular tone by releasing both vasodilating and vasoconstricting substances (Furchtgott
and Zawadsky, 1980; Yanagisawa et al., 1988; Vanhoutte, 1989; Vane et al., 1990). These
mechanisms are important both for the regulation of microvascular (Linder et al., 1990;
Kiowski et al., 1991; Panza et al., 1993; Haynes and Webb, 1994) and larger conduit arteries
(Joannides et al., 1995; Hornig et al., 1996). Patients with chronic heart failure are
hemodynamically characterized by increased vasoconstriction and a reduced vasodilator
response to exercise (Zelis et al., 1968). These abnormalities appear to be due to a number
of compensatory mechanisms and some neurohumoral factors involved in this impaired
vasodilator response have been studied extensively in the past. However, there is growing
evidence now to suggest that the endothelium also plays an important role in the abnormal
vasodilator response. In particular, the role of endothelium-derived nitric oxide has received
considerable attention. The present review, therefore, summarizes the evidence for a reduced
vasodilator capacity of the endothelium in chronic heart failure and focuses on the potential
mechanisms underlying this abnormality. In addition, the importance of endothelial
vasoconstrictor mechanisms is also discussed.
IMPAIRED ENDOTHELIUM DEPENDENT VASODILATION
The endothelium, in response to a variety of stimuli including increased shear stress during
increased blood flow (Joannides et al., 1995; Hornig et al., 1996) or muscarinic receptor
stimulation (Furchgott and Zawadzky, 1980; Linder et al., 1990) releases nitric oxide (NO)
which is formed intracellularly by the action of the enzyme NO-synthase from L-arginine
(Palmer et al., 1987). Following its release and uptake into vascular smooth muscle cells
NO stimulates guanylyl cyclase to form cyclic GMP which then results in smooth muscle
relaxation and vascular dilatation. Because of the ease with which endothelium-dependent,
nitric oxide mediated vasodilator function can be assessed by acetylcholine or other
muscarinic receptor agonists, a number of studies has used regional, e.g. intraarterial
infusions of acetylcholine and measured the resultant vasodilation. Under the assumption
Correspondence: W.Kiowski, MD, Division of Cardiology, University Hospital Zurich, CH-8091 Zurich, Tel:
++41/1/255 23 58; Fax: ++41/1/255 44 01; E-mail: karkiw@unizh.usz.ch
131
132 W.Kiowski and H.Drexler
that a diminished vasodilator response to acetylcholine reflects endothelial dysfunction

(Linder et al., 1990; Panza et al., 1993) evidence for an impaired endothelium-dependent
vasodilator response was found in the microcirculation of the myocardium (Treasure et al.,
1990), leg (Katz et al., 1992), and forearm (Kubo et al., 1991; Drexler et al., 1992; Hirooka
et al., 1992; Katz et al., 1993; Chin et al., 1996). These data are compatible with the view
that the impairment of endothelium-dependent, nitric oxide-mediated vasodilator function
is a generalized phenomenon in patients with chronic heart failure. While the diminished
peripheral vasodilator capacity may be important for the reduced tissue perfusion during
physical exercise and, therefore, is likely to contribute to impaired exercise capacity, the
disturbance of microvascular dilatation in the coronary circulation might result in ischemia
and further myocardial damage and dysfunction in chronic heart failure.
The studies cited so far tested endothelium dependent vasodilator capacity but did not
evaluate the importance of endothelium-derived NO in the maintenance of basal vascular
tone. The regional infusion of N-monomethyl-L-arginine (L-NMMA) a selective inhibitor
of NO-synthase (Moncada et al., 1989) can be used to assess the importance of
endothelium-derived NO for the maintenance of basal vascular tone (Vallance et al., 1989).
Using brachial artery infusions of L-NMMA, it was shown that resistance vessels from
patients with chronic heart failure showed greater vasoconstriction in response to removal
of NO-mediated vasodilation by L-NMMA as compared to control subjects (Drexler et
al., 1992). Therefore, in contrast to stimulated NO-dependent vasodilation, the contribution
of NO to basal tone is preserved or may even be enhanced in forearm resistance vessels
in patients with chronic heart failure (Drexler et al., 1992). However, these results were
obtained in a small group of patients and further studies are needed to better define the
importance of endothelium-derived NO for the regulation of basal vascular tone in heart
failure patients. Interestingly though, peripheral venous nitrate, the product of NO-
metabolism in blood, has also been found to be increased in heart failure (Habib et al.,
1994; Winlaw et al., 1994), a finding compatible with an enhanced basal nitric oxide
production in heart failure.
The study described so far evaluated endothelial function of resistance vessels. Much
less is known about conduit arteries. The measurement by ultrasound techniques (Celermajer
et al., 1993; Joannides et al., 1995; Hornig et al., 1996) of diameter increases of larger
conduit arteries in response to increases in flow as occur e.g. during reactive hyperemia
provides the possibility to study endothelium-dependent vasodilation of larger conduit
arteries in humans. The validity of this approach is shown in Figure 7.1. In seven normal
volunteers, flow-mediated vasodilation was assessed as diameter changes of the radial artery
during hyperemia following upper arm occlusion before and after brachial artery infusion
of the NO-synthase inhibitor L-NMMA (7 µmol/min) (Hornig et al., 1996). As depicted in
the figure, flow-dependent vasodilation (expressed as percent change from baseline vessel
diameter) was signicantly reduced following NO-synthase inhibition. This finding confirms
previous results (Joannides et al., 1995) and demonstrates that flow-mediated vasodilation
indeed is largely NO driven. Using this approach in patients with chronic heart failure it
was shown that flow-dependent vasodilation was significantly attenuated in patients with
chronic heart failure in the radial artery (Hornig et al., 1996). Interestingly, this impairment
was similar in patients with dilated and ischemic cardiomyopathy and was significantly
higher in the non-dominant arm. Importantly, after blockade of NO-synthase by L-NMMA
flow-dependent forearm vasodilation was similar in control subjects and patients suggesting
that the impairment in patients was mostly due to reduced NO-bioavailability.
Endothelial Control of Vascular Tone in Chronic Heart Failure 133
Figure 7.1 Nitric oxide dependcy of flow dependent vasodilation in normal volunteers. Flow dependent vasodilation
was assessed as percent changes of radial artery diameter following release of an upper arm cuff which was
inflated to suprasytolic pressure for 7 minutes in 7 normal volunteers before adn after brachial artery infusion of
L-NMMA (7 µmol/min) to inhibit NO synthesis. As shown, radial artery vasodilation was markedly reduced
during hyperemia following L-NMMA demonstrating that flow dependent vasodilation is to alarge extent NO
driven (Hornig et al., 1996).
Taken together, endothelial dysfunction is not only present in the microcirculation of

patients with heart failure but also in large conduit vessels. Since conduit vessels are more
than just passive conduits (Ramsey and Jones, 1994) it is tempting to speculate that such an
impairment of flow-mediated vasodilation during exercise might lead to increased impedance
to left ventricular ejection and, thereby, contribute to the hemodynamic derangements
characteristic of chronic heart failure. So far, it is not known what constitutes the primary
stimulus for endothelial vasodilator dysfunction in chronic heart failure. Animal experiments
suggest that endothelial dysfunction is a progressive, time-dependent process that probably
plays a minor role early in heart failure. Thus, rats in whom heart failure was induced by
coronary ligation and subsequent myocardial infarction demonstrated no evidence of
endothelial dysfunction at week 1 but a reduced acetylcholine response of thoracic aortic
rings was evident after 4 and 16 weeks as compared to shamoperated control rats (Teerlink
et al., 1993).
MECHANISMS OF ENDOTHELIAL VASODILATOR DYSFUNCTION
A number of factors might be responsible for the impaired NO-dependent vasodilation in

chronic heart failure (Table 7.1) and many of these aspects have been studied in humans.
Table 7.1 Potential mechanisms of reduced acetylcholine responses

in chronic heart failure.
Obviously, the reduced endothelium-dependent, NO-mediated vasodilator response

to acetylcholine may be due to a reduced activity of the NO-forming enzyme NO synthase.
Since pulsatile flow (Rubanyi et al., 1986; Awobsi et al., 1994) and the associated shear
stress (Buga et al., 1991; Uematsu et al., 1995) are important regulators of NO-production
it is tempting to speculate that the reduced cardiac output and stroke volume may be the
link to impaired endothelial vasodilator function in chronic heart failure. Recent
experimental evidence supports this possibility. Thus, in dogs with heart failure after 1
month of rapid ventricular pacing NO-synthase gene expression was reduced by 56% in
endothelial cells from the thoracic aorta as compared to control animals. In addition,
there was a marked, e.g. 70 % reduction in endothelial cell NO-synthase protein in heart
failure dogs and a marked reduction in nitrate production as a measure of enzyme activity
in response to stimulation by either acetylcholine or bradykinin. Interestingly, this down-
regulation of NO-synthase was accompanied by a similar down-regulation of endothelial
cyclooxygenase-1 in heart failure dogs (Smith et al., 1996). Moreover, nitric oxide
production from microvessels isolated from patients with endstage heart failure undergoing
transplantation appeared to be reduced as compared to nitrite production from the
microvasculature of normal hearts (Kichuk et al., 1996). However, the number of normal
hearts was too small to allow a statistically valid comparison. Taking together, these
experimental studies indicate that stimulated endothelial NO-production may be reduced
in patient with chronic heart failure, possibly through reduced gene expression of vascular
endothelial NO-synthase.
Abnormalities found in studies using acetylcholine might also be explained by a defect
at the muscarinic receptor level or its signal transduction pathway. Also, the majority of
patients with heart failure has underlying ischemic heart disease and coronary risk factors
are known to adversely affect the vascular response to acetylcholine (Creager et al., 1990;
Linder et al., 1990; Panza et al., 1993; Seiler et al., 1993); however, a reduced response to
acetylcholine was also found in heart failure patients with non-ischemic cardiomyopathy
and without coronary risk factors (Nakamura et al., 1996). Moreover, acetylcholine has
direct smooth muscle contracting properties (Furchtgott and Zawadsky, 1980). Therefore,
the response to acetylcholine always represents the net effects of the release of vasodilating
substances from the endothelium and direct smooth muscle vasoconstriction. Accordingly,
the use of a pure endothelium-dependent vasodilator without effects on vascular smooth
muscle might provide a more clear picture of endothelial vasodilator capacity. Substance P
is a peptide which stimulates NO-synthase through a different endothelial receptor, e.g. the
tachykinin receptor (Saito et al., 1991) and has no direct effects on smooth muscle cells.
Substance P has been shown to result in dilation of epicardial coronary arteries and coronary
microvessels in humans (Crossman et al., 1989) and of forearm resistance and capacity
vessels (McEwan et al., 1988). This vasodilator effect can be significantly attenuated by
the NO-synthase inhibitor L-NMMA in normal subjects (Panza et al., 1994).
Interestingly, acetylcholine-induced dilation of forearm resistance vessels was
significantly reduced in patients with heart failure whereas the increase in forearm blood
flow in response to substance P was not impaired (Hirooka et al., 1992). Similarly,
intracoronary infusions of acetylcholine caused significantly less increases in coronary blood
flow in heart failure patients as compared to control subjects whereas substance P resulted
and similar increases in coronary blood flow in both groups (Holdright et al., 1994).
Furthermore, the epicardial vasodilator response to substance P was also similar in both
groups (Holdright et al., 1994). These studies, therefore, are compatible with the contention
that chronic heart failure may be associated with a specific receptor abnormality of the
muscarinic receptor and/or post receptor coupling mechanisms which could contribute to
the observed reduction of the response to acetylcholine in patients. These data suggest also
that substance P may be a better pharmacological to investigate endothelial NO-dependent
vasodilation than acetylcholine. Nevertheless, results obtained with stimulation of NO-release
through another, non-muscarinic receptor suggest that the abnormality in heart failure patients
is not likely to be explained solely by a defect at the muscarinic receptor level. Thus,
vasodilation in response to stimulation of vasopressin type-2 (V2) receptors (Hirsch et al.,
1989) is dependent on endothelial NO release and can be blocked by L-NMMA but not
indomethacin (Liard, 1994; Tagawa et al., 1995). When the V2-receptor agonist
desmopressin was infused into the brachial artery of heart failure patients and control subjects
the ensuing vasodilation was significantly attenuated in patients (Rector et al., 1996).
Moreover, inhibition of NO-synthesis by L-NMMA reduced desmopressin responses to a
significantly greater extent in control subjects as compared to patients (Rector et al., 1996).
Accordingly, these data are compatible with the view that impaired endothelium-dependent
vasodilation in patients with chronic heart failure is not limited to a defect of the muscarinic
receptor or its signal transduction pathway.
EFFECTS OF L-ARGININE
In some studies, brachial artery infusions of L-arginine have been shown to augment the
forearm vasodilator response to acetylcholine in normal subjects (Panza et al., 1993; Hirooka
et al., 1994) but this finding is not universal. The effects of intraarterial L-arginine on the
response to acetylcholine have also been studied in patients with heart failure (Hirooka et
al., 1994). L-arginine augmented the vasodilator response to acetylcholine in normal subjects
except for the highest dose. In contrast, in patients with heart failure, L-arginine also
augmented the vasodilator response to the highest dose of acetylcholine. Moreover, L-
arginine did not affect the postischemic increase in forearm blood flow after upper arm
occlusion in normal subjects; however, it significantly increased postischemic blood flow
in patients with heart failure (Hirooka et al., 1994). While the meaning of an enhanced
vasodilator response to only the highest dose of acetylcholine in patients is somewhat
uncertain the finding of an increased maximal vasodilator response after pretreatment with
L-arginine during reactive hyperemia is interesting. These findings may suggest that impaired
endothelium-dependent vasodilation during maximal pharmacological stimulation depends
upon substrate availability/utilization and would be compatible with the view that impaired
post-ischemic vasodilation results largely from a defect in release of nitric oxide from the
endothelium in heart failure patients.
In contrast to results obtained with intraarterial administration of L-arginine, oral
supplementation with L-arginine in doses between 5.6 and 20g/day for 4–6 weeks failed
to augment the response to muscarinic receptor stimulation (Chin et al., 1996; Rector
et al., 1996). Also, it did not enhance reactive hyperemia (Rector et al., 1996). Thus,
oral administration of L-arginine does not suffice to improve endothelial, NO-dependent
vasodilator dysfunction in patients with heart failure. Interestingly though, oral L-
arginine improved functional status as indicated by increased distances during a six
minute walk test and lower scores on the Living With Heart Failure questionnaire in
one (Rector et al., 1996) but not the other study (Chin et al., 1996). Further studies are
needed to identify whether and how oral L-arginine supplementation might affect NO-
dependent vasodilation in patients with heart failure. Appropriate control studies with
D-arginine are also required.
ROLE OF CYTOKINES
Recent data suggest that increased levels of cytokines might be involved in the development
of peripheral endothelial dysfunction in patients with heart failure. Circulating cytokines,
and particularly tumor necrosis factor alpha (TNFα), are increased in severe heart failure
(Wiedermann et al., 1993). Experimental evidence suggests that TNFα decreases constitutive
nitric oxide synthase messenger RNA in vascular endothelial cells by shortening its half-
life (Yoshizumi et al., 1993) and increases expression of the inducible form of nitric oxide
synthase in vascular endothelial (Gross et al., 1991) and smooth muscle cells (Busse and
Mulsch, 1990). Moreover, it increases vascular smooth muscle production of superoxide
anion (O2-) which decreases the half-life of nitric oxide (Matsubara and Ziff, 1986). Thus,
TNFα may either stimulate or inhibit endothelium-dependent, nitric oxide-mediated
vasodilation dependent on the predominant effects of either decreasing NO-bioavailability
through enhanced destruction or increasing NO-bioavailability through NO synthase
induction. So far, there is little data available in humans. Interestingly, TNFα concentrations
were closely correlated with forearm blood flow responses to brachial artery infusions of
acetylcholine (Katz et al., 1994). Thus, even moderate increases in serum TNFα
concentrations as found in that study may be sufficient to activate the inducible form of
nitric oxide synthase and potentiate the vascular effects of constitutive nitric oxide synthase
stimulation by acetylcholine (Katz et al., 1994). A study in human hand veins in vivo showed
that TNFα potently inhibits endothelium-dependent relaxation in these vessels (Bhagat et
al., 1997).
SUPEROXIDE
A further possibility which might explain endothelial dysfunction due to reduced NO-
bioavailability is increased degradation of NOs. Principle among the substances involved
in the breakdown process of NO is O2-, an avid scavenger of endothelium-derived NO
Figure 7.2 Effect of acute and chronic vitamin C administration on NO dependent component of flow mediated
radial artery vasodilation in patients with heart failure. NO dependency was assessed as difference between control
measurements and measurements during NO synthesis inhibition by brachial artery L-NMMA. Placebo
administration did not change the markedly diminished response in heart failure patients but both acute brachial
artery infusion and chronic oral supplementation with vitamin C markely improved the NO dependent component
of flow dependent vasodilation so that it was no longer different from control subjects.
(Gryglewski et al., 1986). Accordingly, prevention of O2- formation or administration of

antioxidant substances which scavenge O2- like vitamin C (Frei et al., 1989) could provide
information for the importance of this potential mechanism. So far, only limited data is
available in patients with heart failure. However, a recent study suggests that increased
oxidative stress may well be an important contributing factor to the impaired endothelial
vasodilator function of patients with chronic heart failure. Thus, as shown in Figure 7.2, the
fraction of flow dependent radial artery vasodilation which is mediated by NO (as assessed
by NO-synthase inhibition by brachial artery infusion of L-NMMA) was markedly impaired
in patients with heart failure as compared to control subjects (Hornig et al., 1998). While
placebo did not change this impaired vasodilator response in patients, acute intraarterial
administration of vitamin C significantly enhanced NO-mediated, flow dependent
vasodilation in patients with heart failure. Moreover, oral administration of vitamin C resulted
in maintenance of this improved vasodilator function (Hornig et al., 1998). Therefore, these
data suggest that increased NO-degradation due to oxidative stress may well be an important
factor contributing to impaired NO-dependent vasodilatation in patients with heart failure.
The primary stimulus for increased oxidative stress remains to be established. However,
increased levels of cytokines and among them TNFα increase O2- release from human
endothelial cells (Matsubara and Ziff, 1986). Also, angiotensin II which is high in advanced
heart failure stimutes vascular O2- production via membrane NADH/NADPH oxidase
activation (Rajagopalan et al., 1996).
GUANYLYL CYCLASE RESPONSIVENESS
NO stimulates guanylyl cyclase in vascular smooth muscle cells and the resultant increase
in cyclic GMP leads to vasodilatation. It is conceivable that a reduced responsiveness of
this system to NO-stimulation could also contribute to endothelial vasodilator dysfunction.
This possibility has been tested in most studies by assessing the vasodilator response to
arterial infusion of a direct NO-donor (e.g. nitroglycerin or sodium nitroprusside) and a
reduced response to nitroglycerin was observed by Zelis et al. in their landmark study of
the peripheral circulation in, 1968 already (Zelis et al., 1968). While many more recent
studies did not find significant differences in the vascular responses to direct-acting NO-
donors the opposite has also been reported. Thus, nitroglycerin resulted in a significantly
smaller increase in mean blood flow velocity of the superficial femoral artery after
intraarterial infusion in patients with heart failure as compared to control subjects (Katz et
al., 1992) and brachial artery infusions of nitroglycerin caused significantly less forearm
resistance vessel dilatation in heart failure patients as compared to control subjects (Katz et
al., 1993). Therefore, a reduced vascular smooth muscle responsiveness to NO-dependent
cyclic GMP-mediated vasodilation may also contribute to this apparent endothelial
vasodilator dysfunction, at least in some patients.
Finally, acetylcholine also stimulates production of endothelium-derived vasoactive
substances originating from the cyclooxygenase metabolic pathway (Katusic et al., 1988)
and an acetylcholine-mediated cyclooxygenase-dependent vasoconstrictor effect has been
reported in a canine model of heart failure (Kaiser et al., 1989). This possibility was tested
in a study of the forearm circulation in which patients with heart failure showed a blunted
response to acetylcholine as compared to control subjects (Katz et al., 1993). When these
experiments were repeated after cyclooxygenase inhibition with indomethacin the vasodilator
response to acetylcholine was unchanged in normal subjects but significantly increased in
patients (39%). Despite this improvement, the response was still significantly attenuated as
compared to normal subjects (Katz et al., 1993). In addition, intraarterial infusion of sodium
nitroprusside in heart failure patients treated with aspirin resulted in significantly greater
vasodilatation as compared to patients not pretreated with aspirin (Jeserich et al., 1995).
Both findings are compatible with the view that an abnormal production of cyclooxygenase
dependent vasoconstricting factor(s) seems to be present in the peripheral circulation of
patients with heart failure. Such an effect may blunt the vasodilatory effects of both
endogenous NO liberated by e.g. acetylcholine as well as of exogenous NO derived from
direct NO donors.
ENDOTHELIN DEPENDENT VASOCONSTRICTION
Endothelin-1 is the principal endothelin isoform of the family of endothelins and it is the
most potent vasoconstrictor substance generated in the human vascular wall (Yanagisawa
et al., 1988). Plasma levels of the peptide are increased 2 to 3 fold in patients with heart
failure, particularly in more advanced heart failure (Cody et al., 1992; Wei et al., 1994;
Kiowski et al., 1995) and the increase in the plasma levels is correlated with the extent of
hemodynamic impairment (Kiowski et al., 1991; Cody et al., 1992). This is shown in
Figure 7.3 which demonstrates significant correlations between plasma levels of this
peptide and cardiac filling pressures, pulmonary pressure, and cardiac output in patients
Figure 7.3 Relationships between baseline plasma endothelin-1 concentrations and the extent of pulmonary
hypertension, cardiac filling pressures and cardiac output in patients with chronic heart failure. The correlations
indicate that higher plasma endothelin-1 concentrations are associated with a greater degree of pulmonary
hypertension, more severely elevated right and left sided cardiac filling pressures and lower cardiac output.
with symptoms according to NYHA class III. Plasma big endothelin-1 which may better
reflect endothelial synthesis than the mature peptide also predicts prognosis in heart failure
(Pacher et al., 1996). Because endothelin-1 has also anti-natriuretic and mitogenic
properties (Ito et al., 1991; Sorensen et al., 1994) it has been suggested that it may play
an important role in the pathophysiology of chronic heart failure. Endothelin exerts its
vascular effects through two principal endothelin receptor subtypes, ETA and ETB. Vascular
smooth muscle ETA receptors mediate endothelin-1-induced vasoconstriction (Riezebos
et al., 1994) and endothelial ETB receptors mediate vasodilation through release of either
prostacyclin and/or nitric oxide (Takayanagi et al., 1991; Hirata et al., 1993). However,
there is also evidence, that vascular smooth muscle ETB receptors mediate vasoconstriction
(Gray et al., 1994; Seo et al., 1994; Haynes et al., 1995). Accordingly, stimulation of
vascular ETB presumably results in a mixture of endothelium-mediated vasodilation and
direct smooth muscle vasoconstriction.
The advent of specific endothelin-receptor antagonists has provided the opportunity
to investigate the role of endothelin-1 mediated vasoconstriction under normal
circumstances and in patients with heart failure. As shown in normal volunteers, infusion
of the ETA receptor antagonist BQ 123 resulted in significant forearm vasodilation
indicating that endothelin is involved in the regulation of basal vascular tone in normal
subjects (Haynes and Webb, 1994).
In patients with heart failure, intravenous infusion of the mixed ETA/ETB receptor
antagonist bosentan also resulted in significant hemodynamic effects (Kiowski et al., 1995).
As shown in Figure 7.4, endothelin receptor antagonism resulted in significant decreases of
left and right heart filling pressures, arterial pressure and pulmonary artery pressure together
with an increase in cardiac output as compared to placebo administration. Since heart rate
did not change, the increase in cardiac output was due to an increase in stroke volume.
Accordingly, calculated systemic vascular resistance was significantly decreased and,
importantly, pulmonary vascular resistance was also significantly reduced. The results
indicate, that blockade of endogenous endothelin-1-mediated vasoconstriction resulted in
significant hemodynamic improvement in patients with advanced heart failure pointing
towards the importance of this endothelial vasoconstrictor system in patients with heart
failure.
So far, the role of the ETB receptor in patients with heart failure is not clear. Thus, the
vasoconstrictor response to a specific ETB receptor agonist, sarafotoxin S6c was increased
in patients with heart failure compatible with an increased sensitivity of this receptor to
endothelin-1 (Love et al., 1996). However, a more recent study using the ETB receptor
antagonist BQ 788 suggested that the overall effect of ETB receptor stimulation in the human
forearm may be dilatation (Verhaar et al., 1998). However, more studies are needed to
ascertain the specific role of ETB receptors in the control of the circulation in patients with
heart failure.
CONCLUSIONS
There is abundant evidence showing that chronic heart failure is associated with impaired
endothelium-dependent microvascular and larger conduit vessel vasodilator dysfunction.
Although much of this impairment can be attributed to reduced NO bioavalability the precise
mechanism(s) underlying this defect remain(s) to be established. The role of endothelin-1-
mediated vasoconstriction and the importance of different endothelin receptors in chronic
heart failure also need further study.
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960–70.
Hypercholesterolaemia is a major risk factor for the development of atherosclerosis. Increasing experimental
evidence suggests that, in common with other risk factors, hypercholesterolaemia may adversely affect endothelial
function. One molecule of particular interest in the prevention of atheroma is the endothelial mediator nitric
oxide. Nitric oxide prevents adhesion of leucocytes to the endothelium, inhibits platelet activation and prevents
smooth muscle cell growth. In the presence of hypercholesterolaemia the bioactivity of endothelium-derived
nitric oxide is reduced and superoxide is generated. Work in our laboratory indicates that increasing nitric oxide
by providing excess L-arginine retards the development of early atheroma in animal models. Furthermore, the
arginine might enhance apoptosis in intimal lesions. Studies in patients have indicated that endothelium-dependent
dilatation is lost in the presence of hypercholesterolaemia and this can be restored by lipid lowering treatment.
Again L-arginine seems to exert a beneficial effect. This might be because it reverses tonic inhibition of nitric
oxide synthase caused by accumulation of the endogenous inhibitor asymmetric dimethylarginine. This chapter
describes the evidence that the nitric oxide pathway is central to the link between hypercholesterolaemia and
atherogenesis in humans.
Key words: Nitric oxide, endothelium, cholesterol, LDL cholesterol, ADMA, superoxide anion, monocytes, plaque
rupture.
8 Hypercholesterolemia, Atherosclerosis, and the NO
Synthase Pathway
John P.Cooke1 and Mark A.Creager2

1
Division of Cardiovascular Medicine, Stanford University, Stanford, CA 94305 5406, USA
2
Vascular Medicine and Atherosclerosis Unit, Brigham & Women’s Hospital and
Harvard Medical School, Boston, MA 02115, USA
CHOLESTEROL AND ATHEROGENESIS

In Europe and the United States, atherosclerosis affecting the coronary or carotid arteries is
the greatest cause of morbidity and annual mortality. Elevated levels of serum cholesterol
correlate with the clinical prevalence of atherosclerosis. Myocardial infarctions are much
less common in countries where dietary intake of cholesterol and serum cholesterol levels
are low, than in countries where individuals consume a Western diet and have higher
cholesterol levels. (Keys, 1980; Kannel, Castelli and Gordon, 1979). Other major risk factors
are hypertension, diabetes mellitus, tobacco use and family history of premature
atherosclerosis. Additional determinants include sedentary state, lipopotein(a), and
hyperhomocysteinemia.
Atherosclerosis has been said to be a “response to injury” of the endothelium. However,
the mechanism(s) by which hypercholesterolemia and the other risk factors alter the
endothelium have not been determined. Recent data from our laboratories and others
indicates that there may be a common pathway by which these risk factors affect endothelial
function. Experimental models of hypertension, hypercholesterolemia, diabetes mellitus or
tobacco exposure, are characterized by common endothelial abnormalities; increased
generation of superoxide anion and reduced bioactivity and/or synthesis of endothelium
derived nitric oxide (Rajagopalan et al., 1996; Ohara, Petersen and Harrison, 1993;
Tesfamariam and Cohen, 1992; Tsao et al., 1995). These aberrations of endothelial function
represent an oxidative stress to the cells and occurs within minutes to hours of exposure to
the noxious stimuli. Oxidative stress perturbs the cell membrane, and increases endothelial
permeability. Moreover, the increased endothelial elaboration of oxygen-derived free radicals
activates an oxidant sensitive transcriptional pathway mediated by nuclear factor ? B(NF?B).
Usually NF?B is found in the cytoplasm in an inactive state, bound to its protein inhibitor
I?Ba (Murohara, et al., 1994; Mercurio et al., 1997). However, oxidative stress activates an
I?B kinase, leading to phosphorylation, ubiquitination and ultimately degradation of I?Ba
(Mercurio, et al., 1997; Woronicz et al., 1997). This leaves NFKB free to translocate to the
nucleus, where it induces the expression of adhesion molecules (eg. vascular cell adhesion
molecule or VCAM-1) that participate in monocyte adhesion and infiltration (Cybulsky
and Gimbrone, 1991; Berliner et al., 1995; Tsao et al., 1996; Tsao et al., 1997; Ross, 1997).
The expression of these adhesion molecules and chemokines may explain the observation
Correspondence: John P.Cooke, MD, PhD, Division of Cardiovascular Medicine, Stanford University School
of Medicine, 300 Pasteur Drive, Stanford, CA 94305–5406, USA.
147
148 J.P.Cooke and M.A.Creager
that within several days of a high cholesterol diet, monocytes adhere to the endothelium,
particularly at intercellular junctions (Ross, 1997).
The monocytes migrate into the subendothelium, where they begin to accumulate lipid
and become foam cells. This is the earliest event in the formation of the fatty streak.
These activated monocytes (macrophages) release mitogens and chemoattractants that
recruit additional macrophages as well as vascular smooth muscle cells into the lesion.
As foam cells accumulate in the subendothelial space they distort the overlying
endothelium, and eventually may even rupture through the endothelial surface (Ross,
1997). In these areas of endothelial ulceration, platelets adhere to the vessel wall, releasing
epidermal growth factor, platelet-derived growth factor, and other mitogens and cytokines
that contribute to smooth muscle migration and proliferation. These factors induce smooth
muscle cells in the vessel wall to proliferate and migrate into the area of the lesion. These
vascular smooth muscle cells undergo a change in phenotype from a “contractile” cell to
a “secretory” cell. These secretory vascular smooth muscle cells elaborate extracellular
matrix (eg. elastin), which transforms the lesion into a fibrous plaque. Extracellular matrix
may contribute significantly to growth of the lesion. A genetic variant of the stromelysin
promoter causes reduced degradation of extracellular matrix (Ye et al., 1996). Extracellular
matrix accumulates and leads to accelerated progression of atherosclerosis (Ye et al.,
1996). The smooth muscle cells may also become engorged with lipid to form foam
cells. The lesion grows with the recruitment of more cells, the elaboration of extracellular
matrix, and the accumulation of lipid until it is transformed from a fibrous plaque to a
complex plaque.
The complex plaque typically is characterized by a fibrous cap which overlies a necrotic
core. The necrotic core is composed of cell debris and cholesterol, and contains a high
concentration of the thrombogenic tissue factor, secreted by macrophages. In later stage
lesions, calcification may occur. Calcifying vascular cells in the vessel wall can transform
into osteoblast-like cells and secrete bone proteins such as osteopontin (Parhami et al.,
1997). Microscopic examination of these areas reveals histology very similar to bone tissue.
Oxidized lipoprotein stimulates the elaboration of bone protein by these vascular cells.
Intriguingly, oxidized lipoprotein reduces bone formation by osteoblasts (Parhami et al.,
1997). This recent finding may account for the clinical observation that some patients with
atherosclerosis (typically elderly women) appear (by X-ray) to have nearly as much calcium
in their aorta as in their spine.
Plaque rupture is the most common cause of myocardial infarctions. Rupture of the
complex plaque exposes the flowing blood to the highly thrombogenic constituents of the
plaque (the foam cells, which elaborate tissue factor). Microscopic examination of the
ruptured plaque generally reveals that the rupture has occurred at the shoulder of the lesion.
In this area the fibrous cap can be seen to be thinned. Immunohistochemical studies reveal
an intense concentration of macrophages in the area, which are elaborating copious amounts
of metalloproteinases (Knox et al., 1997). These macrophages appear to be undermining
the fibrous cap, weakening it and predisposing to its rupture under the stress of hemodynamic
forces. Ruptured plaques generally have a greater macrophage density than stable plaque
(Lendon et al., 1991). The determinants of plaque rupture include the size, eccentricity and
composition of the necrotic core. The larger, more eccentric or more fluid the necrotic
core, the greater the mechanical stress on the fibrous cap (Loree et al., 1994). Fluidity of
the necrotic core is determined by the proportion of cholesterol ester (greater fluidity) and
crystalline cholesterol (less fluidity) contained in the core (Loree et al., 1994).
Hypercholesterolemia, Atherosclerosis, and the NO Synthase Pathway 149
An important determinant of plaque rupture is the thickness of the fibrous cap (Loree et
al., 1994). The fibrous cap, weakened and thinned by the degradative action of the
macrophages, ruptures under the stress of hemodynamic forces. With rupture of the plaque,
the luminal blood comes into contact with highly thombogenic tissue factor deposited by
macrophages in the necrotic core (Marmur et al., 1996). Under the influence of tissue factor,
thrombus forms in the fissures of the lesion. The thrombus often extends into, and may
occlude, the lumen. Plaque rupture and thrombus formation is the most common cause of
heart attack and stroke. Furthermore, as the thrombus organizes it can contribute to growth
of the lesion and increase the symptoms of the patient.
As can be gleaned from the above discussion, plaque growth and rupture is an
inflammatory process, with the participation of vascular adhesion molecules and chemokines,
and monocyte adherence and infiltration. Inflammation of the fibrous cap leads to plaque
rupture. The causative factors initiating inflammation of the fibrous cap are unknown.
However, there is mounting circumstantial evidence that implicates infection in acute
coronary syndromes (Benditt, Barrett and McDougall, 1983; Libby, Egan and Skarlatos,
1997). There is seroepidemiological and immunohistochemical evidence that infectious
agents such as cytomegalovirus, herpes virus, or chlamydia pneumoniae are associated
with atherosclerotic vascular disease and vascular events (Melnick, Adam and DeBakey,
1990; Gratton et al., 1989; Saikku et al., 1988; Thom et al., 1992; Muhlstein et al., 1996;
Minick et al., 1979). Such infections may trigger plaque rupture by increasing hemodynamic
stress (e.g., tachycardia and increased cardiac output that may accompany a febrile illness)
or may directly affect the vascular biology of the plaque. Infection localizing to the plaque
may activate endothelial cells to express adhesion molecules, may stimulate vascular cells
to undergo proliferation, and/or induce resident inflammatory cells to elaborate cytokines
that promote further local inflammation (Libby, Egan and Skarlatos, 1997). Endothelial
cells infected with CMV or herpes virus express leukocyte adhesion molecules which may
participate in monocyte and T-cell recruitment (Sedmark et al., 1995; Etingin, Silverstein
and Hajjar, 1991).
NITRIC OXIDE: AN ENDOGENOUS ANTI-ATHEROGENIC

MOLECULE
As we shall see, endothelium-derived nitric oxide (NO) is a potent inhibitor of the processes
that lead to development of an atherosclerotic plaque. NO is a product of the metabolism
of L-arginine by the endothelial isoform of NO synthase (Moncada and Higgs, 1995).
NO is a potent endogenous vasodilator exerting its actions in the same way as do exogenous
nitrovasodilators such as nitroglycerin. NO released from the endothelium diffuses to the
subjacent vascular smooth muscle and activates soluble guanylate cyclase within the
vascular smooth muscle, leading to the production of cyclic guanosine monophosphate
(cGMP). This cyclic nucleotide is the second messenger for the action of endothelium-
derived NO as well as exogenous nitrovasodilators, and it activates cGMP-dependent
kinases and phosphatases that mediate vascular smooth muscle relaxation. NO is not
only a potent vasodilator but also has important effects on circulating blood elements
and on the vascular wall that may protect the vessel from the development of
atherosclerosis. NO inhibits platelet adherence and aggregation (Radomski, Palmer and
Moncada, 1987; Stamler et al., 1989; Pohl and Busse, 1989). Together, the endothelial
products NO and prostacyclin confer a resistance to platelet-vessel wall interaction. NO

exerts its effect on platelet reactivity in part by stimulating intra-platelet production of
cGMP which subsequently phosphorylates proteins which regulate platelet activation and
adherence (Pohl et al., 1994). Platelets themselves contain small amounts of NO synthase
and are capable of generating NO which may act as a brake on their activation (Radomski,
Palmer and Moncada, 1990; Mehta et al., 1995).
NO also prevents adherence of leukocytes to the endothelium (Kubes et al., 1991).
This salutary effect of NO was first discovered using models of ischemia-reperfiision.
When the coronary artery of an experimental animal is ligated, this induces ischemia of
the myocardium subserved by that vessel. When the ligature is released, the ensuing
reperfusion is associated with further injury to the myocardium which is in part due to
the adherence and infiltration of neutrophils, and the concomitant release of oxygen-
derived free radicals. The adherence of leukocytes and subsequent reperfusion injury can
be markedly inhibited by the simultaneous perfusion of the coronary artery by exogenous
NO donors (Johnson et al., 1990).
Atherosclerosis begins with an alteration in the adhesiveness of the endothelium for
circulating monocytes. The production of NO can be modulated in the laboratory; a reduction
in its synthesis accelerates atherosclerosis, whereas an increase in its synthesis suppresses
and can even reverse atherosclerosis. One explanation for the observation is that NO inhibits
monocyte adherence to the endothelium. Indeed, the ability of monocytes to bind to
endothelial cells in vitro is inhibited by exogenous NO in a dose-dependent manner (Bath
et al., 1991). Moreover, within minutes of exposure to NO, endothelial cells become more
resistant to monocyte adherence (Tsao et al., 1995). Because of the rapid time course, this
effect of NO must be due to inhibition of signaling pathways involved in adhesion-perhaps
by a cGMP-dependent mechanism. More chronic exposure to NO suppresses gene expression
of adhesion molecules (such as VCAM), and chemokines (such as MCP-1) involved in
monocyte adhesion and infiltration. By contrast, inhibition of NO synthesis increases the
expression of endothelial proteins required for monocyte adhesion (Zeiher et al., 1995).
NO appears to exert its effects on gene expression by blocking the activation of specific
transcriptional proteins (such as nuclear factor kB) (Figure 8.1) (Tsao et al., 1996; Tsao et
al., 1997).
Accumulating evidence supports the hypothesis that NO exerts its effect on monocyte
adherence and accumulation in part by modulating the activity of the redox-responsive
transcriptional pathways described earlier (Tsao et al., 1996; Tsao et al., 1997; Marui et
al., 1993; DeCaterina et al., 1995; Spiecker, Peng and Liao, 1997). Vascular cells exposed
to oxidized lipoprotein or cytokines begin to elaborate superoxide anion (Tsao et al., 1996;
Tsao et al., 1997). This generation of reactive oxygen species is associated with the
transcription of a number of genes participating in atherogenesis including MCP-1 mRNA.
MCP-1 increases chemotactic activity of the vessel wall for monocytes (Tsao et al., 1997).
These effects are all suppressed by the NO-donor, DETA-NO. NO may reduce the half-life
of MCP-1 mRNA, as well as reducing its transcription. NO exerts its effects on MCP-1
expression in cytokine-stimulated HUVECs in a cGMP-independent fashion.
It is well established that hypercholesterolemia reduces the bioactivity of endothelium-
derived NO (Heistad et al., 1984; McLenahan et al., 1991). (see below). In parallel, the
endothelium begins to generate superoxide anion (Ohara, Petersen and Harrison, 1993).
This increased endothelial generation of superoxide anion induced by hypercholesterolemia
in rabbit models can be reversed by placing the animal on a low cholesterol diet (Ohara et
Figure 8.1 Atherosclerotic risk factors such as hypercholesterolemia, hypertension, tobacco, and diabetes mellitus
lead to increased free radical production and decreased nitric oxide activity in endothelial cells. This endothelial
dysfunction not only has acute effects on vascular tone, but also chronic effects on vessel structure. Increased
superoxide anion leads to activation of NFkB via phosphorylation and degradation of the inhibitor protein
IKBa. NFKB is then free to translocate into the nucleus to initiate transcription of proatherogenic genes such
as VCAM-1 and MCP-1. Nitric oxide can inhibit these processes by inhibiting superoxide production, directly
scavenging superoxide anions, as well as increasing the transcription and activity of I?Ba. Moreover, since NO
is a paracrine factor, it can have important inhibitory effects on circulating leukocytes and underlying smooth
muscle cells.
al., 1995). The reduction in superoxide anion generation is associated with an improvement
in endothelium-dependent vasodilator function. In addition to inducing the generation of
superoxide anion, hypercholesterolemia causes a decline in tissue glutathione levels, and
thereby increases susceptibility to oxidative damage (Ma et al., 1997). This alteration in
endothelial redox state triggers the oxidant-sensitive transcriptional cascade that results in
the activation of genes encoding molecules that regulate endothelial adhesiveness (Tsao et
al., 1996; Marui et al., 1993). The cytokine-induced activation of VCAM-1 and MCP-1 in
cultured endothelial cells is suppressed by antioxidants or NO donors (Tsao et al., 1996;
Tsao et al., 1997). This effect of NO appears to be due in part to stabilization and/ or
increased expression of I?Ba, which complexes with NF?B to inhibit its transcriptional
activity (Peng, Libby and Liao, 1995; Spiecker, Peng and Liao, 1997).
NO may act by reducing intracellular oxidative stress. There are several possible
mechanisms by which NO may reduce oxidative stress. NO can scavenge superoxide anion,
although the product of this reaction, peroxynitrite anion, is itself a highly reactive free
radical (Radi et al., 1991). However, it is possible that peroxynitrite anion could subsequently
nitrosylate sulfhydryl groups to form S-nitrosothiols (Radi et al., 1991). This class of
molecules is known to induce vasodilation, inhibit platelet aggregation, and interfere with
leukocyte adherence to the vessel wall (Stamler et al., 1992). Another mechanism by which
NO may ameliorate oxidative stress is by terminating the autocatalytic chain of lipid
peroxidation that is initiated by oxidized LDL or intracellular generation of oxygen-derived
free radicals. Indeed, exogenous NO inhibits copper-induced oxidation of LDL cholesterol,
causing a lag in the formation of conjugated dienes (Hogg et al., 1993). Finally, NO may
directly suppress the generation of oxygen-derived free radicals by nitrosylating, and thereby
inactivating oxidative enzymes. This hypothesis is supported by the observation that the
generation of superoxide anion by stimulated neutrophils is reduced by their exposure to
exogenous NO (Clancy et al., 1992). This is due to the inactivation of NADPH oxygenase,
a multimeric enzyme, with cytosolic and particulate components. The particulate component
is vulnerable to nitrosylation by NO (either at its heme moiety or sulfhydryl group), which
prevents its association with the cytosolic component, and reconstitution of the active
enzyme. A similar phenomenon may occur in endothelial cells. This would explain the
observation of Niu and colleagues, that antagonism of endogenous NO production increases
oxidative stress in HUVECs, as demonstrated using redox-sensitive fluorophores (Niu, Smith
and Kubes, 1994). Furthermore, Pagano and colleagues (1993) have shown that exogenous
NO donors inhibit the generation of superoxide anion by the endothelium of rabbit thoracic
aortae treated ex vivo with antagonists of superoxide dismutase. Thus, NO appears to exert
its effects, in part, by reducing intracellular oxidative stress, thereby defusing oxidant-
triggered transcription.
NO also regulates the growth of vascular smooth muscle cells. In vitro, NO-donors inhibit
the proliferation of vascular smooth muscle cells; this effect is mimicked by exogenous
administration of 8-bromo-cGMP, a stable analog of the second messenger of NO action
(Garg et al., 1989). Other agents such as atrial natriuretic peptide which increase the
intracellular levels of cGMP inhibit proliferation of vascular smooth muscle cells in culture.
Does NO inhibit the proliferation of vascular smooth muscle cells in vivo? Some initial
studies indicate that NO does indeed play an important role in controlling vascular growth.
In experimental animal models, chronic inhibition of NO synthesis causes hyperreactivity
to vasoconstrictors, and medial thickening, in the coronary microvasculature (Numaguchi
et al., 1995; Ita et al., 1995). These effects are not mediated by the hypertension that is
induced by NOS antagonists, because co-administration of hydralazine to normalize blood
pressure does not reverse the effects of NOS antagonists upon microvasculature structure
and function (Numaguchi et al., 1995) In a number of disease states where the release of
NO is reduced or abolished, such as restenosis, hypercholesterolemia, and hypertension,
there is an increase in the proliferation of vascular smooth muscle cells within the media
and the intima. In experimental animal models, augmentation of endogenous NO synthesis
(as with administration of the NO precursor L-arginine), inhibits “restenosis” (myointimal
hyperplasia) after balloon angioplasty; the effect of L-arginine is blocked by antagonists of
NO synthase (McNamara et al., 1993; Tarry and Makhoul, 1994). In one study, after
subjecting the carotid artery to balloon angioplasty, the vessel was transfected with a plasmid
construct containing the gene encoding NO synthase. This gene transfer had the effect of
enhancing NO synthesis locally and inhibiting myointimal hyperplasia (von der Leyen et
al., 1995). Also, intramural administration of a single dose of L-arginine, at the time of
balloon angioplasty, can markedly inhibit myointimal hyperplasia 2–4 weeks later
(Schwarzacher et al., 1997). This effect of L-arginine is associated with increased local
production of NO, probably due to utilization of L-arginine by induced NO synthase in
vascular smooth muscle cells in the injured area.
Proliferation of vascular smooth muscle cells, as well as monocyte adherence and
infiltration, platelet adherence, and aggregation, are key processes involved in atherogenesis.
Because endothelium-derived NO inhibits each of these processes, we have proposed that
NO is an endogenous anti-atherogenic molecule (Cooke et al., 1992; Cooke and Tsao,
1993; Cooke and Tsao, 1994; Cooke and Dzau, 1997). Therefore an endothelial injury or
alteration which results in a reduction in NO activity could promote atherogenesis.
EFFECT OF NO RESTORATION ON ATHEROGENESIS
If a reduction in NO activity promotes atherogenesis, a restoration of NO activity might be

expected to retard progression of the disease. Cooke and colleagues tested the hypothesis
that increased synthesis of NO by the vessel wall would inhibit atherosclerosis. New Zealand
White rabbits were placed on normal or high cholesterol diet; some of the animals on the
high cholesterol diet also received supplemental dietary arginine or methionine to increase
the intake of these amino acids six-fold (Cooke et al., 1992; Wang et al., 1994). After 10
weeks, the thoracic aortae and coronary arteries were harvested for studies of vascular
reactivity and histomorphometry. Supplemental dietary arginine did not alter the lipid profile
nor any hemodynamic parameters in the hypercholesterolemic animals; the only difference
between the hypercholesterolemic groups was that plasma arginine was doubled in the
hypercholesterolemic animals receiving dietary arginine supplementation. As expected, NO-
dependent vasodilation to acetylcholine was inhibited in the hypercholesterolemic animals
receiving vehicle. By contrast hypercholesterolemic animals receiving L-arginine had an
improvement in NO-dependent vasodilation. The improvement of NO-dependent
vasodilation in hypercholesterolemic animals receiving L-arginine was associated with a
striking effect on vascular structure. Chronic L-arginine administration reduced the surface
area of the thoracic and abdominal aorta involved by lesions in the hypercholesterolemic
animals (Figure 8.2) (Cooke et al., 1992). In the left main coronary artery the differences
were even more striking; no lesions at all were observed in the hypercholesterolemic animals
receiving L-arginine (Wang et al., 1994). The mechanism by which dietary arginine inhibits
atherogenesis appeared to be due, in part, to an inhibition of monocyte-endothelial cell
interaction in the hypercholesterolemic animals. Boger and colleagues have shown that
administration of L-arginine to hypercholesterolemic rabbits increases vascular NO
production, and reduces superoxide anion elaboration (1995). As mentioned previously,
the balance between nitric oxide and superoxide anion may have profound effects on the
activation of genes regulating monocyte adhesion and accumulation. Indeed, after exposure
to a high cholesterol diet the thoracic aorta of hypercholesterolemic animals elaborates
more superoxide anion (Ohara, Petersen and Harrison, 1993; Boger et al., 1995) and has
increased adhesiveness for monocytes (Tsao et al., 1994). Compared with the thoracic aortae
from normal animals, those from hypercholesterolemic rabbits manifested a three-fold
increase in the number of adherent cells. The increase in cell binding is attenuated in thoracic
aortae from hypercholesterolemic animals receiving dietary arginine supplements (Figure
8.3) (Tsao et al., 1994). This is associated with an increase in the release of NO from these
tissues, as well as a reduced vascular expression of MCP-1 (Tsao et al., 1996). The number
Figure 8.2 Photomicrographs of abdominal aorta from New Zealand White rabbits fed a high cholesterol diet in
the absence (top panel) or the presence (bottom panel) of supplemental dietary arginine.
of adherent cells is dramatically increased in normocholesterolemic animals receiving the

NO synthase antagonist nitro-arginine (Figure 8.3) (Tsao et al., 1994). The increased
adhesiveness of the endothelium in the nitro-arginine treated animals is associated with
increased vascular expression of MCP-1 (Tsao et al., 1996). Thus, reductions in NO activity
by hypercholesterolemia, or inhibition of NO synthesis, is associated with increased
monocyte-endothelial cell binding, possibly due to the increased expression of specific
chemokines and adhesion molecules. Indeed, a number of investigators have now shown
that administration of NO synthase antagonists accelerates atherosclerosis in animal models
(Cayette et al., 1994; Naruse et al., 1994; Aji et al., 1997).
Apoptosis has been reported to occur in vascular cells of human atherosclerotic plaque
(Isner et al., 1995; Geng and Libby, 1995). Administration of arginine restores NO activity
in hypercholesterolemic rabbits, and is associated with regression of pre-existing lesions
(Candipan et al., 1996). Preliminary studies indicate that enhancement of vascular NO
activity causes regression by inducing apoptosis of macrophages within the lesion. Factors
involved in the initiation and regulation of apoptosis in atherosclerosis have not been
fully elucidated, but immunohistochemical studies provide evidence for several proteins
known to participate in apoptosis, including p53 (Bennett, Evan and Schwarz, 1995).
Among the myriad pathways that may be involved, there is accumulating evidence to
implicate L-arginine/NO synthase (Messmer, Lapetina and Brune, 1995). Cytokine-
mediated activation of iNOS induces peroxynitrate generation and apoptosis of
macrophages in vitro (Ischiropoulos, Zhu and Beckman, 1992; Lin et al., 1995). The
effect of iNOS activation is augmented by additional L-arginine, and attenuated by
antagonists of NO synthase. Therefore it is possible that enhancement of vascular NO
protection could induce apoptosis and regression of pre-existing lesions. Accordingly, in
a study by Wang et al, male New Zealand White rabbits were fed a 0.5% cholesterol diet
for 10 weeks and subsequently placed on 2.5% L-arginine HCl in the drinking water. The
cholesterol diet was continued for two weeks, at which time the aortae were harvested
for histological studies. As observed by Hoechst staining, L-arginine treatment increased
the number of apoptotic cells (largely macrophages) in the intimal lesions by three-fold
(Wang et al., in press). In subsequent studies, aortae were harvested for ex vivo
examination. Aortic segments were incubated in cell culture medium for 4 to 24 hours
with modulators of NO synthase pathway. The tissues were then collected for histological
studies, and the conditioned medium collected for measurement of nitrogen oxides by
chemiluminescence. Addition of sodium nitroprusside to the medium caused a time-
dependent increase in apoptosis of vascular cells (largely macrophages) in the intimal
lesion. L-arginine (10-3 M) had an identical effect on apoptosis, which was associated
with an increase in NO released into the medium. These effects were not mimicked by D-
arginine, and they were antagonized by the NO synthase inhibitor, L-nitro-arginine (10-4
M). The effect of L-arginine was not influenced by an antagonist of cGMP-dependent
protein kinase, nor was the effect mimicked by the agonist of protein kinase G or 8 Br
cyclic GMP. These results indicated that supplemental L-arginine induces apoptosis of
macrophages in intimal lesions by its metabolism to nitric oxide, which acts via a cyclic-
GMP independent pathway. Previous studies have suggested that apoptosis induced by
iNOS activity may be mediated in part by mechanisms independent of cGMP (Bennett,
Evan and Schwarz, 1995; Messmer, Lapetina and Brune, 1995). Furthermore, these studies
explain a previous observation that supplementation of dietary arginine induces regression
of atheroma in this animal model (Candipan et al., 1996). It is likely that iNOS expressed
Figure 8.3 Histograms illustrating the results of an ex vivo functional binding assay. New Zealand White rabbits
were fed a high cholesterol or normal chow diet. Some hypercholesterolemic animals received oral L-arginine
supplementation, whereas some normocholesterolemic animal received L-nitro arginine (the NOS antagonist) in
their drinking water. After two weeks the thoracic aortae were harvested. In an ex vivo functional binding assay
with monocytoid cells, the thoracic aortae of hypercholesterolemic animals had greater adhesiveness for monocytoid
cells, than did the aortae from normocholesterolemic animals (top panel). L-arginine supplementation reduced
endothelial adhesiveness. By contrast the administration of L-nitro arginine to normocholesterolemic animals
markedly increased the adhesiveness of their aortae (bottom panel).
by cells within the lesion is responsible for the effect of L-arginine. Indeed, previous
immunohistochemical studies have detected iNOS in the intimal macrophages and vascular
smooth muscle cells of human atherosclerotic plaque (Buttery et al., 1996). These are
activated cells which also produce superoxide anion. In this milieu, the product of iNOS is
quickly transformed into peroxynitrite anion, a highly reactive free radical. Peroxynitrite
anion is cytotoxic and induces apoptosis (Ischiropoulos, Zhu and Beckman, 1992; Lin et
al., 1995). Peroxynitrite anion can also affect cell function by nitrosylating tyrosine residues
that are involved in the signal transduction of transmembrane receptors. Using monoclonal
antibodies directed against nitrotyrosine, evidence of peroxynitrite formation has been
observed in human atherosclerotic plaque (Beckman et al., 1994). This is relevant to the
study described above, since peroxynitrite anion is likely the NO species mediating the
apoptosis observed in the aforementioned study.
The activation of iNOS may have complex effects on the evolution of atherosclerotic
plaque. By inducing cell death, iNOS activation may contribute to the development of the
“necrotic core” of complex lesions. One might also speculate that iNOS may be involved in
the characteristic atrophy of the media beneath atheroma or the dissolution of the fibrous
cap by activated macrophages, as peroxynitrite anion or other NO donors may induce
apoptosis of vascular smooth muscle (Pollman et al., 1996) Furthermore, peroxynitrite anion
may reduce collagen formation by vascular cells, and activate metalloproteinases which
degrade extracellular matrix (Rajagopalan et al., 1996). These actions of peroxynitrite anion
would contribute to plaque instability and have led some to explore antagonism of iNOS as
a potential therapeutic avenue. However, it is likely that such a strategy would have
unintended consequences. Antagonism of iNOS activity could promote platelet aggregation,
leukocyte adherence, vasoconstriction, and proliferation of vascular smooth muscle cells
and macrophages. We speculate that iNOS may be in fact a countervailing force in the
accretion of atherosclerotic plaque. Furthermore, by reducing proliferation and by promoting
apoptosis of macrophages in the lesion, iNOS activation may lead to plaque stabilization
and even regression. It is worthy of emphasis that both macrophages and vascular smooth
muscle cells contribute to the intimal lesion in the balloon-injured hypercholesterolemic
rabbits, but it was largely the macrophages that appear to undergo apoptosis in an animal
model (Wang et al. in press). The contrarian concept that iNOS may act as a brake on
vascular inflammation and lesion development has recently received strong experimental
support. Rat aortic allografts develop significant increases in intimal thickness associated
with an increase in inducible NO synthase expression (Shears et al., 1997). Inhibition of
NO synthase activity (using a NO antagonist) or expression (using cyclosporine), increases
intimal thickening. By contrast, adenoviral-mediated iNOS gene transfer abolishes allograft
vasculopathy in this model (Shears et al., 1997). This report is consistent with the notion
that the product of iNOS activation may suppress recruitment of inflammatory cells, reduce
their proliferation and/or enhance their apoptosis to act as a countervailing force in vascular
inflammation.
EFFECT OF HYPERCHOLESTEROLEMIA ON NITRIC OXIDE
The mechanism whereby hypercholesterolemia causes atherosclerosis may be mediated,

in part, by its effect on the availability and subsequent activity of endothelium-derived
NO. The bioavailability of endothelium-derived NO can be assessed in experimental
models and in humans by administering pharmacologic agents that result in activation of

eNOS and by assessing the vasorelaxant response of arteries in vitro or vasorelaxation in
vivo. Endothelium-dependent relaxation, mediated by nitric oxide, is abnormal in arteries
isolated from hypercholesterolemic animals (Verbeuren, 1986; Cohen et al., 1988;
Bossaller et al., 1987). Incubation of normal aortic rings with LDL cholesterol, particularly
modified or oxidized LDL cholesterol, blunts endothelium-dependent relaxation (Andrews
et al., 1987; Jacobs, Plane and Bruckdorfer, 1990; Takahashi et al., 1990; Simon,
Cunningham and Cohen, 1990). A specific component of oxidized LDL,
lysophosphatidylcholine, inhibits endothelium-dependent relaxation when it is placed in
the incubation medium (Yokoyama et al., 1990; Kugiyama et al., 1990; Flavahan, 1993).
Also, endothelium-dependent vasodilation to parenteral administration of acetylcholine
is reduced in peripheral resistance vessels of hypercholesterolemic models in vivo (Osborne
et al., 1989; Girerd et al., 1990). Thus, abnormal endothelium-dependent vasodilation
occurs in vascular preparations exposed to cholesterol and in vessels of
hypercholesterolemic animals, even in the absence of atherosclerosis. These observations
are consistent with the premise that reduced availability of NO in hypercholesterolemic
states precedes, and may contribute to, atherosclerosis.
Endothelium-dependent vasodilation is abnormal also in hypercholesterolemic humans.
Creager et al. (1990) reported that methacholine-induced, endothelium-dependent
vasodilation is impaired in forearm resistance vessels of hypercholesterolemic patients who
have no clinical manifestations of atherosclerosis. (Figure 8.4). These observations have
been confirmed by others (Celermajer et al., 1992; Casino et al., 1993; Stroes et al., 1995).
Hypercholesterolemia has been shown to impair endothelium-dependent vasodilation in
coronary arteries, even in the absence of atherosclerosis (Zeiher et al., 1991). Moreover,
the cholesterol concentration correlates with the vasoconstrictive response to acetylcholine
in epicardial coronary arteries of patients with coronary atherosclerosis (Vita et al., 1990).
There even appears to be an inverse relationship between cholesterol and endothelium-
dependent vasodilation among healthy subjects whose cholesterol levels are less than 200
mg/dl (Steinberg et al., 1997).
Conversely, lipid lowering therapy improves endothelial function in experimental
models and in patients with hypercholesterolemia. Harrison et al. (1987) found that dietary
restriction of cholesterol restored endothelium-dependent relaxation of atherosclerotic
iliac arteries excised from cynomolgus monkeys previously fed a high cholesterol diet.
In humans, lipid lowering therapy has been reported to improve endothelium-dependent
vasodilation of coronary and peripheral arteries. Six months of treatment with
cholestyramine has been shown to enhance endothelium-dependent vasodilation of
angiographically normal epicardial coronary arteries of hypercholesterolemic patients
(Leung, Lau and Wong, 1993). Similarly, HMG Co-A reductase inhibitors, administered
for six months improved endothelium-dependent vasodilation of atherosclerotic coronary
arteries of hypercholesterolemic patients (Egashira et al., 1994; Treasure et al., 1995).
Also, HMG Co-A reductase inhibitors administered for one to three months, have been
reported to improve endothelium-dependent vasodilation in forearm resistance vessels (Stroes
et al., 1995; Leung, Lau and Wong, 1993; O’Driscoll, Green and Taylor, 1997). Even acute
reduction of cholesterol with LDL apheresis has been shown to improve endothelium-
dependent vasodilation in the forearm of patients with hypercholesterolemia, within hours
of treatment (Tamai et al., 1997). Thus, reduction of cholesterol appears to restore the
Figure 8.4 The effect of hypercholesterolemia on endothelium-dependent vasodilation. The forearm blood flow
response to the endothelium-dependent vasodilator, methacholine choloride, was less in hypercholesterolemic
subjects than in age-matched healthy subjects. Adapted from Creager et al. (ref. 99).
vasoactive regulating component of endothelial function, in vessels with and without

atherosclerosis, and it might do so within a short period of time.
Multiple mechanisms may account for the abnormalities in the L-arginine/NO pathway
induced by hypercholesterolemia. These include: reduced availability of L-arginine;
abnormalities of endothelial receptor-G protein coupling; decreased levels of cofactors such
as tetrahydrobiopterin; reduced nitric oxide synthase expression and activity; increased
degradation of NO by superoxide anion or oxidized lipoproteins or, a circulating inhibitor
of NO synthase (Cooke and Dzau, 1997).
L-arginine and Asymmetric Dimethylarginine

Several years ago we found that L-arginine, but not D-arginine improved endothelium-
dependent vasodilation to acetylcholine in the hindlimb of cholesterol fed rabbits (Girerd
et al., 1990). We further observed that endothelium-dependent relaxation from thoracic
aortae excised from cholesterol-fed rabbits treated with L-arginine is greater than those
from rabbits treated with vehicle (Cooke et al., 1991). Subsequently, we studied
hypercholesterolemic humans and found that acute parenteral administration of L-arginine,
but not D-arginine, improves endothelium-dependent vasodilation of forearm resistance
vessels (Creager et al., 1992). L-arginine also acutely improves endothelium-dependent

vasodilation of coronary resistance vessels of hypercholesterolemic humans Drexler et al.,
1991). Clarkson et al. (1996) reported that L-arginine administered orally for four weeks,
improved flow-mediated endothelium-dependent vasodilation of the brachial artery of
hypercholesterolemic patients.
The mechanism by which L-arginine exerts its beneficial effects is likely through enhanced
production of endothelium-derived NO. Until recently it was not apparent how exogenous
L-arginine could increase NO elaboration in hypercholesterolemia, given the low Km of the
purified endothelial NO synthase (2.9 µM) (Pollock et al., 1991), and the relatively high
physiological plasma concentrations of L-arginine (60–100 µmol/L), which are not reduced
in hypercholesterolemia. This “L-arginine paradox” may be due, in part, to the presence of
endogenous competitive NOS inhibitors like asymmetric dimethylarginine(ADMA) (Figure
8.3). Recently, ADMA has been characterized to be an endogenous, competitive inhibitor
of NO synthase (Vallance et al., 1992). ADMA has been shown to be synthesized by human
endothelial cells (Fickling et al., 1993). The plasma level of ADMA is elevated in
hypercholesterolemic rabbits (Bode-Boger et al., 1996). as well as in hypercholesterolemic
and atherosclerotic humans (Boger et al., 1997; Boger et al., in press) concomitantly with
impaired endothelial NO elaboration. ADMA plasma levels, which are 1.0±.0.1 µmol/L in
healthy humans, are elevated to 2.2±.0.2 µmol/L in hypercholesterolemic individuals (Boger
et al., 1997). In elderly patients with peripheral arterial disease and generalized
atherosclerosis, ADMA levels range from 2.5 to 3.5 µmol/L, corresponding to the severity
of the vascular disease (Boger et al., 1997). Cooke and colleagues have examined the effect
of ADMA upon cultured endothelial cells (Boger et al., under review). Incubation of
endothelial cells with ADMA (at concentrations that are observed in hypercholesterolemic
humans) inhibit NO production. This effect is associated with increased endothelial
superoxide radical elaboration and NFkB activation, resulting in enhanced MCP-1 expression
and endothelial adhesiveness for monocytes. These effects of ADMA are reversed by L-
arginine.
Intracellular ADMA levels within endothelial cells may be higher than those values
observed in plasma. Support for this speculation comes from studies of regenerated
endothelium. After balloon angioplasty and endothelial denudation, the endothelial
monolayer regenerates. However, these cells are morphologically abnormal, displaying a
polygonal, cuboidal appearance; poorly developed intercellular junctions, with occasional
gaps between endothelial cells; and a lack of the usual alignment with the flow field
(Shimokawa, Aarhus and Vanhoutte, 1987; Weidinger et al., 1990; Weidinger et al., 1991).
Furthermore, endothelium mediated vasodilation is impaired in vessels with a regenerated
endothelium (Shimokawa, Aarhus and Vanhoutte, 1987; Weidinger et al., 1990; Weidinger
et al., 1991). A recent study has shed light on the latter observation. Isolation of endothelial
cells from the luminal lining that has regenerated after balloon injury of the rabbit iliac
artery, reveals intracellular levels of ADMA that are three-fold higher than those observed
in endothelial cells from uninjured vessel (Azumi et al., 1995).
The observation that endothelial cells are a source of ADMA corroborates the previous
finding by Fickling et al. (1993) that human endothelial cells release ADMA and SDMA.
ADMA competitively inhibits endothelial NO synthesis in an autocrine manner. This
observation is in line with previous findings that exogenous ADMA concentrations between
1 and 10 µM affect the activity of the NO synthase in the vasculature of rat mesentery
tissue and rat brain (Kurose et al., 1995; Faraci, Brian and Heistad, 1995). Faraci et al.
(1995) calculated an IC50 value for the inhibition of NO production in rat cerebellar
homogenate by ADMA of 1.8±.1 µM, and Fickling et al. (1993) reported that 2 and 10 µM
ADMA inhibited nitrite production in LPS-simulated J774 macrophages by 17 and 33%
respectively. Taken together, these data suggest that ADMA may be a potential autocrine
regulator of endothelial NO synthase.
The source of ADMA in endothelial cells is currently unclear. Dimethylarginines are
likely the result of degradation of methylated proteins (McDermott, 1976; MacAllister et
al., 1996). The specific enzyme S-adenosylmethionine: protein arginine N-methyltransferase
(protein methylase I) has been shown to methylate internal arginine residues in a variety of
polypeptides, yielding NG-monomethyl-L-arginine, NG, NG-dimethyl-L-arginine, and NG,
NG,-dimethyl-L-arginine upon proteolysis. The metabolism of ADMA, but not SDMA, occurs
via hydrolytic degradation to citrulline by the enzyme dimethylarginine
dimethylaminohydrolase (DDAH) (MacAllister et al., 1994). Inhibition of DDAH causes a
gradual vasoconstriction of vascular segments, which is reversed by L-arginine (MacAllister
et al., 1996). This latter finding suggests that regulation of intracellular ADMA levels affects
NO synthase activity. Intriguingly, Cooke and colleagues have developed preliminary data
indicating that low-density lipoprotein increases endothelial cell ADMA elaboration.
Although the mechanism by which LDL may affect ADMA formation or metabolism is
currently unknown, this finding suggests that elevated ADMA levels may mediate some of
the effects of LDL on the endothelium in an autocrine manner. Most importantly, as described
above, within the concentration range found in cultured endothelial cells (5–40 µM), ADMA
can induce some of the pathophysiological changes of the endothelium that occur in
hypercholesterolemia.
G Protein Receptor Coupling, NO Synthase and Cofactors

Hypercholesterolemia may interfere with G protein endothelial receptor coupling, thereby
inhibiting initial steps in signal transduction that ultimately activate NO synthase. Native
LDL reduces bradykinin stimulated production of NO by inhibiting guanine nucleotide
binding proteins which couple the receptor to activation of NO synthase (Liao and Clark,
1995). Hypercholesterolemia may impair endothelium-dependent relaxation of porcine
coronary artery rings via a pertussis toxin-sensitive, Gi protein-dependent pathway
(Shimokawa and Vanhoutte, 1989). Flavahan (1993) found that lysophosphatidylcholine,
which is associated with oxidized LDL, inhibited endothelium-dependent relaxation induced
by serotonin, but not bradykinin or ADP, in porcine coronary rings indicating that
lysophosphatidylcholine selectively inhibits a Gi protein-dependent pathway. In contrast,
Tanner et al. (1991) reported that oxidized LDL caused endothelial impairment which could
be reversed by L-arginine.
Tetrahydrobiopterin (BH4) is a cofactor for NO synthase and is involved in the electron
transfer required for synthesizing NO from L-arginine (Gorren et al., 1996; Klatt et al.,
1992). BH4 increases production of NO by eNOS and decreases the generation of superoxide
anion(Wever et al., 1997). Rabelink and colleagues suggested that relative depletion of
BH4 may contribute to endothelial dysfunction since intra-arterial administration of BH4
improved endothelium-dependent vasodilation to serotonin in hypercholesterolemic patients
(Stroes et al., 1997).
Liao et al. (1995) reported that oxidized LDL, but not native LDL, decreases levels of
NO synthase mRNA. This effect of oxidized LDL on expression of eNOS is mediated via
inhibition of transcription as well as an increase in post-transcriptional degradation (Liao et

al., 1995).
Oxidant Stress
As noted previously, NO is inactivated by superoxide anion, generating peroxynitrite
(Gryglewski, Palmer and Moncada, 1986; Mugge et al., 1991; Rubanyi and Vanhoutte, 1986).
Superoxide anion production is increased three to five-fold in cholesterol-fed rabbits compared
to control rabbits (Ohara, Petersen and Harrison, 1993; Roger et al., 1995). Chin et al. (1992)
found that the lipid component of oxidized LDL inactivated NO released from bovine aortic
endothelial cells. In addition, hypercholesterolemia impairs glutathione-mediated detoxification
of peroxynitrite anion (Ma et al., 1997). This reduces NO regeneration from peroxynitrite
and also makes vascular tissue more susceptible to oxidative injury.
In animal models of hypercholesterolemia, antioxidant treatment with either superoxide
dismutase, oxypurinol, a-tocopherol or probucol, restores endothelium-dependent relaxation
implicating degradation of NO by superoxide anion as a cause of endothelial dysfunction
(Ohara, Petersen and Harrison, 1993; Mugge et al., 1991; White et al., 1994; Keaney et al.,
1994; Keaney et al., 1995; Levine et al., 1996). Comparable observations have been made
in hypercholesterolemic humans. The addition of probucol, a lipid lowering drug with
antioxidant properties, to lovastatin improved endothelium-dependent vasodilation of
epicardial coronary arteries in patients with hypercholesterolemia and coronary artery disease
to a greater extent than the combination of lovastatin and cholestyramine despite similar
reduction in LDL cholesterol (Anderson et al., 1996). This difference was attributed to the
antioxidant properties of probucol since improvement in endothelial function correlated
with prolongation of the lag phase of copper-induced LDL oxidation cholesterol (Anderson
et al., 1996). Antioxidant treatment with vitamin C also improves endothelium-dependent
vasodilation in hypercholesterolemic patients in peripheral vessels devoid of atherosclerosis.
Vitamin C is a water soluble antioxidant that is capable of scavenging superoxide anion.
Ting et al. (1997) found that acute intra-arterial administration of vitamin C enhanced the
endothelium-dependent vasodilator response to methacholine chloride in
hypercholesterolemic subjects (Figure 8.5). Similarly, Levine et al. (1996) reported that
oral ingestion of vitamin C acutely improved flow-mediated endothelium-dependent
vasodilation of the brachial artery of hypercholesterolemic patients with coronary artery
disease. Taken together, there is compelling evidence that superoxide anion production is
increased in hypercholesterolemia and contributes importantly to reduced bioavailability
of endothelium-derived nitric oxide and subsequent reduction of endothelium-dependent
vasodilation. The oxidative enzymes that are responsible for endothelial secretion and
superoxide anion may include xanthine oxidase, NADPH oxygenase, or even NO synthase,
under certain conditions (e.g., arginine depletion). NOS may itself generate superoxide
anion (Pou et al., 1992; Pritchard et al., 1995; Huk et al., 1997), an observation that may
explain both the beneficial effects at anti oxidants and those of L-arginine.
SUMMARY
Vascular disease begins with an alteration in the endothelium which is characterized by an

increase in intracellular oxidative stress, and the activation of oxidant-response genes
Figure 8.5 The effect of the antioxidant, vitamin C, on endothelium-dependent vasodilation in

hypercholesterolemic subjects. Vitamin C enhanced the forearm blood flow response to methacholine, implicating
oxidant stress, i.e., superoxide anion, as a cause of endothelial dysfunction in hypercholesterolemia. Adapted
from Ting et al. (ref. 153).
regulating the expression of adhesion molecules and chemokines. These changes promote
interaction of the endothelium with circulating blood elements. Monocyte infiltration and
foam cell formation ensue, followed by further endothelial dysfunction and damage which
precipitates platelet adherence and proliferation of vascular smooth muscle. These key
processes in atherogenesis are opposed by nitric oxide. NO suppresses the expression and
signaling of adhesion molecules involved in monocyte adhesion to the vessel wall, and
inhibits platelet adherence and vascular smooth muscle cell proliferation. The NO synthase
pathway is perturbed by hypercholesterolemia and other metabolic disorders that predispose
to atherosclerosis. It is likely that basic insights regarding the mechanisms of endothelial
dysfunction will lead to new therapeutic strategies to halt the progression, or induce
regression, of atherosclerosis.
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vasodilators (e.g. nitrovasodilators). A drawback of this approach, partially overcome by using selective inhibitors
of nitric oxide (NO) synthase (cNOS) and cyclooxygenase (COX), is that the overall blood flow response to
agonists such as acetylcholine is mediated by nitric oxide (NO), vasoactive prostanoids, endothelium-derived
hyperpolarising factor (EDHF) plus other factors. In type 1 diabetes, especially in the presence of
microalbuminuria, NOS inhibition produces less of an effect on basal blood flow and the acetylcholine stimulated
blood flow response is less readily inhibited by NOS inhibition than in control subjects. This suggests decreased
basal and stimulated release or increased inactivation of NO in type 1 diabetes. The overall response to
acetylcholine may be preserved suggesting that there is a compensatory increase in vasodilator COX products
or EDHF. In type 2 diabetes vasodilator responsiveness to acetylcholine is impaired but the pathway responsible
has not been clearly elucidated. In both type 1 and type 2 diabetes there is evidence of impaired sensitivity of
vascular smooth muscle to nitrovasodilators suggesting impaired sensitivity to endothelium-derived NO.
Umbilical vein endothelial cells obtained from mothers with gestational diabetes or those from non-diabetic
mothers exposed to elevated concentrations of glucose exhibit an increase in NO and, disproportionately, in
superoxide anion (O2-) production. The latter, through inactivation of NO, might account for the apparent decrease
in NO seen in in vivo studies. Other mechanisms which may account for decreased availability of NO in
diabetes include other sources of free radicals (including O2-) which may inactivate NO, and direct inactivation
of NO by advanced glycosylation end products and/or by oxidised LDL. Improvements in endothelial function
have been seen after lipid lowering therapy in type 1 diabetes and after acute administration of vitamin C in
type 2 diabetes. Other promising strategies include novel antioxidants, angiotensin converting enzyme inhibitors,
angiotensin II receptor antagonists and agents which increase insulin sensitivity.
Key words; diabetes, EDHF, cyclooxygenase, pregnancy, arginine transport, lipoproteins.

9 Endothelial Function in Diabetes Mellitus
Jim M.Ritter1,2, Phil J.Chowienczyk1,2 and Giovanni E.Mann2

1
Department of Clinical Pharmacology, St Thomas’ Hospital, UMDS, Lambeth Palace
Road London, SE1 7EH, UK
2
Centre for Cardiovascular Biology and Medicine, GKT, School of Biomedical
Sciences, King’s College London, Campden Hull Road, W8 7AH, UK
INTRODUCTION
Diabetes mellitus is defined by a fasting blood glucose concentration≥7.0 mmol/L or an

abnormal rise in blood glucose following an oral load. The two commonest forms are type
1 (insulin dependent) diabetes, which has a strongly inherited predisposition and variable
annual incidence in different countries (~1 to 2 per 100,000 in Japan, >40 per 100,000 in
Finland), and type 2 (non-insulin dependent) diabetes, which also has a strong inherited
component and is much commoner than type 1 (Wolff, 1993). Type 1 diabetes is caused by
an autoimmune destruction of the islets of Langerhans following an environmental insult
(e.g. infection with Coxsackie virus) in genetically predisposed individuals. Patients with
type 1 diabetes have a near total loss of insulin secretion, which results in increased glycogen
metabolism, elevated circulating plasma glucose levels and ketoacidosis. Type 2 diabetes is
more complex and in these patients hyperglycaemia is associated with varying degrees of
insulin resistance and relative impairment of insulin secretion (Jaap et al., 1994). Insulin
secretion declines with age and thus type 2 diabetes is increasing in our ageing population.
In contrast to untreated type 1 diabetes, where circulating concentrations of insulin are low
or absent, the plasma concentrations of insulin in type 2 diabetics may in the early stages of
the disease be higher than in non-diabetic subjects. However, blood glucose is elevated due
to impaired responses to insulin either at or beyond the level of the insulin receptor.
Gestational diabetes constitutes another type of diabetes, recognized during pregnancy,
and is generally associated with a transient decrease in maternal insulin sensitivity similar
to that found in patients with non-insulin dependent diabetes (Dornhorst & Beard, 1993;
Eriksson, 1995). Patients with gestational diabetes have elevated levels of hemoglobin A1c
(a marker of protein glycation in diabetes) during early pregnancy (see Sobrevia & Mann,
1997). Such women generally include a small number with previously unrecognized diabetes
or impaired glucose tolerance (Dornhorst & Beard, 1993). Treatment with diet and/or insulin
has been highly successful in reducing the incidence of fetal macrosomia in gestational
diabetic pregnancies.
Both type 1 and type 2 diabetes are associated with vascular complications involving
small and large blood vessels. Microvascular complications of retinopathy, nephropathy
and neuropathy are recognised by clinicians as specific for diabetes mellitus, but it is the
Current Addresses: Professor Jim M.Ritter and Dr Phil J.Chowienczyk, Department of Clinical Pharmacology,
St. Thomas’ Hospital, 9KT, School of Biomedical Sciemces, KCL, Lambeth Palace Road, London SE1 7EH,
UK. Tel: 0171-928 9292 ext. 2350; Fax: 0171-401-2242.
173
174 J.M.Ritter et al.
consequences of macrovascular atheromatous disease, notably myocardial infarction and

thrombotic stroke, that are principally responsible for the increased incidence of disability
and premature death in these patients. Microalbuminuria is the earliest evidence of
glomerular endothelial injury and is a strong risk factor for myocardial infarction in
diabetic patients. The increased thromboxane synthesis in type 2 patients (Davi et al.,
1990) implies that physiological mechanisms involved in preventing platelet activation
may be defective in such patients. Although this contrasts with type 1 patients in whom
abnormal thromboxane production is not a general feature (Green et al., 1988), platelets
from type 1 and type 2 diabetic patients have a significantly reduced activity of NO
synthase and an impaired ability to mediate vasodilation (Oskarsson et al., 1996; Martina
et al., 1998; Rabini et al., 1998). Plasma levels of von Willebrand factor (vWf) are elevated
in patients with diabetic microangiopathy but are apparently not influenced by changes
in plasma glucose, insulin, or growth hormone levels per se (Porta et al., 1981). The
possibility that increased circulating vWf levels may precede microalbuminuria in patients
with insulin-dependent diabetes has been used as indicator for ensuing diabetic
complications (Stehouwer et al., 1995). Moreover, advanced glycosylated end products
(AGEs) are clearly implicated in endothelial cell dysfunction in diabetic patients (Makita
et al., 1991; Bucala et al., 1991), although modulation of the secretory pathways in
endothelial cells remains to be elucidated.
The recognition that atherosclerosis is initiated by endothelial injury, and that the
endothelium synthesises vasoactive factors that protect against progression of atherosclerotic
disease has led to investigations of endothelial function in patients with diabetes. There
have been three main approaches: in vivo studies of vascular responses in diabetic patients,
studies using biopsies of small resistance arteries mounted in organ chambers and investigated
in vitro, and in vitro studies of cultured endothelial cells. Each of these approaches has its
limitations, and one of our objectives is to discuss these critically. Although our current
understanding is fragmentary and incomplete, there is overwhelming evidence based on
these complementary techniques that endothelial function is altered in diabetes mellitus.
We have focused on human studies of the L-arginine/NO signalling pathway in view of its
role in regulating basal vascular tone and the progression of atherosclerosis (Ignarro, 1990;
Moncada et al., 1991). We refer the reader to recent comprehensive reviews of endothelial
dysfunction in experimental animal models of diabetes (Cohen, 1993; Poston & Taylor,
1995; Sobrevia & Mann, 1997).
In vivo Studies of Endothelium Derived Mediators

The role of the endothelium in diabetes mellitus has been examined in vivo mainly by
assessing local vasodilator responses to physical or pharmacological stimuli assumed to
increase endothelium derived mediators. Strain gauge plethysmography (Benjamin et al.,
1995) has been widely used to measure limb blood flow responses to brachial or femoral
artery infusion of endothelium-dependent and -independent vasodilators. This method
reflects the function of resistance vessels, the calibre of which determines limb blood flow.
Forearm vasculature has been extensively studied because of its accessibility, but it is also
of particular interest as it is rarely affected by atheroma (Ciccone et al., 1993), facilitating a
distinction between functional abnormalities and structural effects of early atherosclerosis.
Muscarinic agonists such as acetylcholine and methacholine have been used extensively
and assumed to act largely through the L-arginine/NO pathway (Vallance et al., 1989).
Endothelial Function in Diabetes Mellitus 175
However, it is likely that these agonists also stimulate release of endothelium-derived

hyperpolarizing factor (Chen et al., 1988; Taddei et al., 1995), synthesis of vasoactive
prostanoids (Shimizu et al., 1993; Taddei et al., 1997) and may have endothelium-
independent actions on vascular smooth muscle mediated either directly or through
stimulation of sensory nerves (Ralevic et al., 1992). The relative contributions of these
various actions to the overall vasodilator effect is likely to differ for individual muscarinic
agonists. Actions of acetylcholine are more readily inhibited by the NO synthase inhibitor
NG-monomethyl-L-arginine (L-NMMA) than are those of methacholine, suggesting that
acetylcholine is a more sensitive probe of the NO pathway (Chowienczyk et al., 1993).
However, due to its extreme instability in blood, responses to acetylcholine are highly
dependent on forearm geometry and basal forearm blood flow (Chowienczyk et al., 1994).
Brachial artery infusion of L-NMMA has been used to assess basal NO production, with
the assumption that this is directly related to the degree of forearm vasoconstriction produced
by L-NMMA (Vallance et al., 1989). Use of an appropriate comparator vasoconstrictor,
which does not interact with the L-arginine/NO pathway, may strengthen this approach
(Calver et al., 1992).
A non-invasive method for measuring endothelial function in the brachial artery has
been developed by Celermajer and colleagues (Celermajer et al., 1992). High resolution
duplex ultrasound is used to image the brachial artery and measure changes in artery diameter
in response to changes in flow. Increases in blood flow generated by reactive hyperaemia in
the hand after a short period of radial arterial occlusion (ie distal to the site where the artery
diameter is being measured) are followed by an increase in brachial artery diameter. This
flow-mediated dilation is due, in part, to endothelium-derived NO, since it is attenuated by
prior infusion of L-NMMA into the brachial artery (Joannides et al., 1995). The technique
is capable of detecting subtle degrees of endothelial dysfunction in young subjects
(Celermajer et al., 1993, 1996; Sorensen et al., 1994).
Doppler fluximetry has been used to measure skin blood flow responses to local heating
or iontophoresis of vasoactive drugs (Jaap et al., 1994; Morris et al., 1995). The role of
endothelin in diabetes has been studied mainly by measuring concentrations of endothelins
in plasma and urine. Responses to local infusion of endothelin-1 have been studied in type
2 diabetes. Studies involving local infusion of endothelin receptor antagonists and converting
enzyme inhibitors may provide more specific information.
Type 1 Diabetes
Both basal and agonist-stimulated NO release has been investigated in forearm vasculature
of patients with type 1 diabetes. Calver et al. (1992) reported a blunted response to L-
NMMA in type 1 patients, which was confirmed by Elliott et al. (1993), who in addition
reported that this response was most marked in patients with microalbuminuria. Stimulated
responses have been examined using the muscarinic agonists acetylcholine, methacholine
and carbachol. In most studies vasodilator responses to these agonists were preserved (Halkin
et al., 1991; Calver et al., 1992; Elliott et al., 1993; Smits et al., 1993). However, in patients
with type 1 diabetes, other groups have reported a blunted response to acetylcholine and
methacholine (Mäkimattila et al., 1996; Johnstone et al., 1993). A potentially important
difference in the study by Johnstone et al. (1993) was that subjects were pretreated with
aspirin to inhibit cyclooxygenase, thereby inhibiting the synthesis of vasoactive prostanoids.
This may reconcile their findings with those of Elliott et al (1993), who found that although
Figure 9.1 Synthesis of endothelium derived mediators. Pathways implicated in the pathogenesis of diabetic
angiopathy are shown in bold. Elevated glucose and abnormal lipoproteins may increase generation of from
cNOS. COX activity may be increased but how the balance of vasodilator/vasoconstrictor products is altered in
humans is unknown. The influence of diabetes on EDHF synthesis is unknown; it may be increased. ACh:
acetylcholine; AGEs: advanced glycosylation end products; cAMP: cyclic adenosine monophosphate; cGMP:
cyclic guanylate monophosphate; cNOS: constitutive nitric oxide synthase; COX: cyclooxygenase; EDHFs:
endothelium derived hyperpolarising factors; NO: nitric oxide; : superoxide anion; ox-LDL: oxidised low density
lipoprotein; PG: prostaglandin; ROS: reactive oxygen species.
vasodilator responses to carbaehol were preserved, the NO dependent component of the

response (identified by co-infusion of L-NMMA) was reduced in patients with
microalbuminuria (Figure 9.1). Taken together the results of these studies suggest the
possibility that type 1 diabetes is associated with a reduced NO mediated vasodilatory
response to muscarinic agonists and compensatory increase in vasodilatory prostanoids.
Further studies need to compare the vasodilatory responses to muscarinic agonists in the
absence and presence of cyclooxygenase inhibitors in type 1 patients and controls.
Alternatively, the normal vasodilator response to carbaehol but loss of inhibition by L-
NMMA that were observed could be due to increased co-release of endothelium-derived
hyperpolarising factor (EDHF) by muscarinic agonists in type 1 patients with endothelial
dysfunction.
The apparently contradictory findings in other studies could be the consequence of
differences in patient characteristics. The presence of microalbuminuria and the degree of
glycaemic control are important variables (Halkin et al., 1991; Elliott et al., 1993; Smits et
al., 1993; Mäkimattila et al., 1996). Methodological factors relating to choice of agonist,
especially the relative magnitudes of the NO-and EDHF-mediated components, may also
account for the contrasting findings in studies which have employed methacholine rather
than acetylcholine (Smits et al., 1993; Mäkimattila et al., 1996; Johnstone et al., 1993).
The influence of basal blood flow may be especially important in type 1 diabetes, since
basal flow increases with poor metabolic control. Flow-mediated dilation is impaired in
patients with type 1 diabetes, especially in patients with microalbuminuria (Zenere et al.,
1995), and an inverse association between flow-mediated dilation and LDL-cholesterol has
been described in patients with type 1 diabetes (Clarkson et al., 1996).
Plasma concentrations of endothelin-1 have been reported to be lower in children with
type 1 diabetes than in age matched controls and to be negatively associated with the duration
of diabetes (Smulders et al., 1994; Malamitsi-Puchner et al., 1996). Elevated ET-1
immunoreactivity has been identified in the cutaneous microvasculature of patients with
type 1 diabetes of recent onset, yet ET-1 immunoreactivity is reduced in patients with diabetes
of longer duration and in patients with retinopathy (Properzi et al., 1995).
Insulin Resistance and Type 2 Diabetes

Most studies in type 2 diabetes have focused on stimulated NO release and, unlike type 1
diabetes, there is general consensus that responses to endothelium-dependent agonists
are impaired. McVeigh et al. (1992) first reported impaired forearm blood flow responses
both to acetylcholine and GTN in patients with type 2 diabetes. Reactive hyperaemic
forearm blood was similar in diabetic patients and non-diabetic control groups. Impaired
vasodilator responses to acetylcholine and nitroprusside have also been described in the
skin of type 2 diabetic patients (Morris et al., 1995). These findings were recently extended
by Williams et al (1996), who excluded patients with hypertension and
hypercholesterolaemia, both potentially confounding factors. In this latter study of forearm
blood flow, subjects were pretreated with aspirin to inhibit the endogenous production of
vasoactive prostaglandins. Forearm blood flow responses to both methacholine and
nitroprusside were significantly attenuated in diabetic patients compared with controls,
with no significant group differences noted in responses to verapamil or hyperaemia.
The use of aspirin suggests that abnormal synthesis of vasoconstrictor prostanoids cannot
explain this aspect of vascular dysfunction in type 2 diabetes. Normal responses to
verapamil exclude a non-specific impairment of vasodilator function. A reservation
regarding the interpretation of this study is the use of methacholine, however, similar
results have recently been reported using acetylcholine as an agonist (Watts et al., 1996).
The similarity of altered vascular responses to acetylcholine and methacholine suggests
that EDHF release in response to muscarinic agonists may be impaired in subjects with
type 2 diabetes, and begs a direct comparison of acetylcholine responses in type 2 diabetic
patients in the absence or presence of L-NMMA. This was done by Watts et al., (1996)
who found that L-NMMA produced similar blunting of the response to acetylcholine in
diabetics and controls, thus indirectly implicating EDHF. The study of Watts et al. (1996)
excluded patients on oral hypoglycaemic drugs and those with autonomic neuropathy
and/or proteinuria. Thus, drug therapy and autonomic dysfunction are unlikely to explain
the impaired vasodilator responsiveness in type 2 diabetes, and this defect is not confined
to patients with proteinuria. The impaired dilator responses to GTN or nitroprusside, as
well as to acetylcholine or methacholine, in these studies again raises the question of

what fractions of the observed responses to different muscarinic agonists are mediated
by NO. The findings could be explained by an increased inactivation of NO and/or EDHF,
resulting from an increase in endothelium-derived superoxide anion (O2-) production as
discussed below, or an impaired sensitivity of vascular smooth muscle to NO.
Impaired vasodilator responses to NO donors such as nitroprusside or GTN in type 2
diabetic patients have not been reported by all groups. Impaired forearm vasodilator
responses to methacholine, with preserved responses to nitroprusside, have recently been
reported in type 2 diabetics (Ting et al., 1996). Goodfellow et al. (1996) observed impaired
flow-mediated dilation of the radial artery in type 2 patients, with endothelium-independent
responses to GTN unaffected. Skin vasodilator responses to iontophoresis of acetylcholine
are impaired in patients with type 2 diabetes, yet responses to nitroprusside are apparently
impaired only in patients with peripheral sensory neuropathy (Pitei et al., 1997). We have
also observed impaired endothelium-dependent vasodilation, with preserved responses to
nitroprusside, in forearm vasculature of patients with type 2 diabetes of recent onset without
evidence of peripheral sensory neuropathy (data not shown). One explanation is that well-
controlled type 2 diabetes of recent onset is associated with an impaired release of
endothelium-derived NO (or possibly endothelium-derived hyperpolarizing factor, EDHF),
and that diabetes of longer duration results additionally in increased inactivation of exogenous
NO and/or impaired sensitivity of vascular smooth muscle to NO. This explanation is
supported by findings in patients with insulin resistance who had not developed type 2
diabetes at the time of study. Steinberg et al (1996) measured leg blood flow responses to
femoral artery infusions of methacholine and nitroprusside in insulin sensitive controls,
insulin-resistant obese subjects and patients with type 2 diabetes. The response to
methacholine, but not nitroprusside, was decreased in obese and diabetic subjects.
Euglycemic hyperinsulinemia increased the leg blood flow response to methacholine in
control subjects, whereas responses remained blunted in obese and type 2 diabetic groups.
This important study suggests that insulin-resistant obese subjects exhibit vascular
dysfunction and are resistant to the potentiating effect of insulin on methacholine-induced
vasodilation. It further suggests that these effects are not simply related to changes in blood
glucose concentration.
Baron and colleagues had previously suggested that an impaired vasodilator action of
insulin accounts for insulin resistance (Laakso et al., 1990). In skeletal muscle, vasodilator
actions of insulin are mediated in part by NO (Scherrer et al., 1994). Thus, an impairment
in endothelium-dependent vasodilation could contribute to insulin resistance by reducing
delivery of insulin and glucose to skeletal muscle and hence reducing glucose uptake.
Evidence against this elegant hypothesis is the observation that, in healthy volunteers, local
infusion of L-NMMA attenuates the vasodilator response to insulin without reducing insulin-
stimulated glucose uptake (Scherrer et al., 1994). However it is possible that impaired
endothelium-dependent vasodilation exacerbates insulin resistance by this mechanism in
genetically predisposed subjects, in whom insulin and glucose delivery to skeletal muscle
may be more critical in determining glucose uptake.
An exciting finding is that, as in hypercholesterolaemia (Ting et al., 1996), impaired
responsiveness to methacholine in type 1 and type 2 diabetic patients is corrected by acute
administration of vitamin C (Ting et al., 1996b; Timimi et al., 1998). Vitamin C is likely to
be acting by scavenging free radicals, and this finding therefore suggests the possibility
that increased production of free radicals such as superoxide anions (O2-) may explain the
blunted response to methacholine in type 2 diabetic patients. Dietary supplementation with

fish oil also improves endothelium-dependent vasodilation in type 2 diabetes but the
mechanism of action remains unresolved (McVeigh et al., 1994). Ongoing trials with orally
administered antioxidants suggest that vitamin E is ineffective (Gazis et al., 1998). In contrast,
water soluble antioxidants may prove to be more effective (Ting et al., 1996ab; Timimi et
al., 1998).
Plasma concentrations of immunoreactive endothelin-1 have been reported to be
elevated in patients with type 2 diabetes complicated by retinopathy (Morise et al., 1995;
Patino et al., 1994), but other investigators have found no significant difference between
controls and patients with and without complications (Veglio et al., 1993; Bertello et al.,
1994). Vasoconstriction to endothelin-1 is impaired in men with type 2 diabetes (Nugent
et al., 1996). Thus, if impaired sensitivity to endothelin-1 does have a causal role in the
pathogenesis of type 2 diabetes, it most likely results from hyperperfusion and subsequent
microvascular damage (Nugent et al., 1996). Alternatively, reduced responsiveness to
exogenous ET-1 in type II diabetes may reflect receptor down regulation secondary to
increased endothelin concentrations. Studies with endothelin antagonists should help
resolve these issues.
Mechanisms of Endothelial Dysfunction Suggested by Studies In Vivo

Potential causes of endothelial dysfunction that may be of relevance in either type 1 or
type 2 diabetes include: lipid abnormalities, increased oxidative stress and advanced
glycosylation end products (AGE) (Figure 9.2) (see Cohen, 1993; Poston and Taylor,
1995; Tribe and Poston, 1996; Sobrevia and Mann, 1997). The clinical studies described
above are consistent with increased oxidative stress and inactivation of endothelium-
derived NO or EDHF by free radicals such as O2- (Rubanyi and Vanhoutte, 1986). Oxidative
stress results from an increased generation of free radicals and/or diminished cellular
antioxidant defences (Halliwell, 1993). Free radical generation may be increased in
diabetes as a result of glucose-induced activation of cyclooxygenase, autoxidation of
glucose, increased levels of angiotensin II or an alteration in transition metal metabolism
(Hawthorne et al., 1989; Wolff, 1993; Ceriello et al., 1993; Halliwell, 1993; Tribe and
Poston, 1996; Graier et al., 1996). Increased flux through the polyol pathway can deplete
NADPH which is required for the glutathione-redox cycle (Cohen, 1993; Poston and
Taylor, 1995; Sobrevia and Mann, 1997), and O 2 - generation is increased in
hypercholesterolaemia and possibly in other forms of dyslipidaemia that are associated
with type 2 diabetes, and it is possible that qualitative abnormalities of lipoproteins underlie
endothelial dysfunction in diabetes (McNeill et al., 1998).
Endogenous antioxidants include the enzymatic antioxidants superoxide dismutase
(SOD), catalase and glutathione peroxidase and the non-enzymatic antioxidants vitamins E
and C, all of which may be depleted in type 2 diabetes (Sundaram et al., 1996). Reliable
measures of oxidative stress in vivo have hitherto been lacking (Morrow and Roberts, 1996),
hampering direct investigation of these hypotheses. Lipid peroxidation is a central feature
of oxidant injury, and the recent discovery that this is assocated with non-enzymatic
formation of isoprostanes (prostaglandin-like compounds) has led to the measurement of
isoprostanes as an index of oxidative stress in vivo (Morrow and Roberts, 1996; Roberts
and Morrow, 1997; Patrono and FitzGerald, 1997). F2-isoprostanes have recently been shown
to be elevated in patients with non-insulin dependent diabetes (Gopaul et al., 1995).
Figure 9.2 Effect of the NO synthase inhibitor L-NMMA on the forearm blood flow response to brachial artery
infusion of the endothelium-dependent vasodilator carbachol in healthy control subjects and insulin-dependent
diabetic patients (IDDM) with (+) and without (-) microalbuminuria. Results are the percentage increase in blood
flow ratio (infused: non-infused arm) caused by carbachol 2.5 µg min-1 during co-infusion of saline and L-NMMA.
Values are means±SEM. Adapted from Elliott et al., 1993.
Inactivation of NO and possibly other endothelium-derived mediators such as EDHF by

reactive oxygen radicals provides a plausible and attractive explanation for the ability of
vitamin C to improve vascular responsiveness to muscarinic agonists in acute experiments.
Effects of Diabetes on Relaxation of Isolated Human Blood Vessels In Vitro

Endothelium-dependent relaxation of resistance arteries from women with gestational
diabetes is impaired compared to vessels isolated from normal pregnancies (Knock et al.,
1997). As relaxation to acetylcholine was similar in normal and diabetic arteries in the
presence of indomethacin, these authors concluded that gestational diabetes inhibited the
synthesis of dilator prostaglandins. The sensitivity of arteries to acetylcholine was reduced
to a similar degree by the NO synthase inhibitor NG-monomethyl-L-arginine in normal
pregnant and gestational diabetic women, suggesting that NO synthesis was not impaired
in gestational diabetes. Moreover, gestational diabetes had no effect on the sensitivity of
vascular smooth muscle cells to the NO donor nitroprusside, indicating that NO-mediated
vasodilator responses in maternal resistance vessels were not altered.
Similar studies using resistance arteries from human gluteal fat biopsies have shown
that maximal contractile responses to noradrenaline, potassium and angiotensin II were
attenuated in arterial rings from insulin-dependent diabetic patients (McNally et al., 1994).
Although endothelium-dependent relaxation to acetylcholine was impaired by diabetes,
vasodilator responses to another endothelium-dependent agonist, bradykinin, and to

nitroprusside, were unaffected suggesting a defect in activation/coupling of the
acetylcholine receptor rather than a decreased ability to synthesize and release NO. Recent
studies with human saphenous vein rings have confirmed that endothelium-dependent
relaxation mediated by NO is similar in rings from healthy controls or from either type 1
or type 2 diabetic patients, whereas prostanoid- and fibrinogen-mediated relaxation was
abolished in rings from diabetic patients (Hicks et al., 1997). Thus, impaired endothelium-
dependent relaxation was attributed to a decreased prostacyclin and increased
vasoconstrictor prostanoid synthesis in diabetic patients. Interestingly, this study also
reported that vascular relaxation in response to the Ca2+ ionophore A23187 was reduced
in saphenous vein rings from diabetic patients, consistent with previous findings that
A23187-stimulated release of NO and stored Ca2+ are diminished in endothelial cells
exposed to hyperglycemia (Wascher et al., 1994).
The reactivity of human internal mammary artery and saphenous vein rings isolated
from coronary artery disease patients with type 2 diabetes undergoing coronary artery
bypass surgery is abnormal (Karasu et al., 1995). In contrast to diminished constrictor
responses in human gluteal resistance arteries from type 1 diabetic patients (McNally et
al., 1994), maximal contractile responses to noradrenaline and ET-1 were found to be
increased in internal mammary artery and saphenous vein rings from coronary artery
patients with type 2 diabetes (Karasu et al., 1995). Endothelium-dependent relaxation of
internal mammary artery rings was decreased in coronary artery patients with type 2
diabetes, whereas relaxation to nitroprusside was unaffected. Thus, NO-mediated
relaxation of vascular smooth muscle cells in isolated arterial rings in vitro does not appear
to be impaired in diabetes (McNally et al., 1994; Karasu et al., 1995; Knock et al., 1997;
Hicks et al., 1997).
As described above, qualitative abnormalities in lipoproteins occur in patients with
diabetes mellitus (McNeill et al., 1998) Studies in vitro demonstrate that low density
lipoproteins (LDL), and particularly oxidised LDL, powerfully inhibit endothelium-
dependent relaxation (Jacobs et al., 1990) and agonist-stimulated release of NO and
prostacyclin (Jay et al., 1997). In diabetes, LDL is composed of smaller, denser particles
than LDL in non-diabetic subjects (Austin and Edwards, 1996). Such small dense particles
are more susceptible to oxidation (Austin et al., 1988; Tribble et al., 1992), and indeed
patients with type 2 diabetes have increased plasma antibody levels to oxidised LDL and
concentrations of lipid peroxides, including 8-epi-prostaglandins (Gopaul et al., 1995;
Sundaram et al., 1996). It is possible that the altered characteristics of lipoproteins,
particularly the presence of small dense LDL in diabetes, are responsible for the increased
generation of .
Diabetes-induced Alterations in NO and Prostacyclin Synthesis in Cultured

Endothelial Cells
Micro- and macrovascular endothelial cells exhibit a differential responsiveness to

insulin, with a much higher sensitivity detected in microvascular cells (from calf retina)
than in macrovascular endothelial cells isolated from calf aorta (King et al., 1983).
Human endothelial cells in culture exhibit only a small increase in nucleic acid synthesis
in response to insulin, in contrast to the marked effects of endothelial cell growth factors
(King et al., 1983). Human umbilical vein endothelial cells from type 1 diabetic mothers
function abnormally in vitro, with altered cell membrane lipid composition and abnormal
morphology (Cester et al., 1996), an increased rate of cell proliferation, reduced
resistance to shear stress and lower rates of glucose transport compared to cells isolated
from non-diabetic pregnancies (Sank et al., 1994). In contrast, umbilical vein endothelial
cells, derived from diet-controlled gestational diabetic pregnancies, exhibit decreased
rates of proliferation, elevated intracellular Ca 2+ levels and a sustained membrane
hyperpolarization (Sobrevia et al., 1995). Basal and histamine-stimulated synthesis of
prostacyclin (and PGE2) is markedly reduced in umbilical vein endothelial cells from
gestational diabetic pregnancies (Karbowski et al., 1989; Sobrevia et al., 1995), whereas
basal rates of NO production are increased (Sobrevia et al., 1995). The sustained
membrane hyperpolarization and elevated levels of intracellular Ca2+ in endothelial cells
from gestational diabetic pregnancies may explain the increased basal rates of NO
production (Sobrevia et al., 1996, 1998). Although histamine evokes maximal rates of
NO production in fetal endothelial cells from gestational diabetic pregnancies,
sustained exposure to high glucose inhibits agonist-stimulated NO release (Sobrevia
et al., 1995, 1998).
Studies in fetal endothelial cells from normal pregnancies have established that
elevated glucose (EC50 11 mM) induces a time- (3–6 h) and protein synthesis dependent
increase in the activity and expression of constitutive NO synthase (eNOS) (Sobrevia
et al., 1996; Mann et al., 1998). Similar studies in human aortic endothelial cells have
confirmed that elevated glucose (5 days) increases the release of NO and superoxide
anions by 40% and 300%, respectively, perhaps explaining the imbalance in NO and
O2- production in diabetes (Cosentino et al., 1997). Longer-term exposure (15 days) to
elevated glucose apparently does not alter the expression of NO synthase in human
umbilical vein endothelial cells (Mancusi et al., 1996), suggesting an adaptation in NO
synthesis under conditions of sustained hyperglycemia. Although human insulin (0.1–
1 nM) stimulates eNOS activity in umbilical vein endothelial cells (Sobrevia et al.,
1996), activation of the L-arginine/NO pathway by insulin is markedly attenuated by
elevated glucose and gestational diabetes (Sobrevia et al., 1998). Perhaps the activation
of eNOS by insulin in non-diabetic endothelial cells is a consequence of stimulation of
the pentose cycle by insulin, which would increase the supply of NADPH required for
NO synthesis (Wu et al., 1994).
Our findings in human umbilical vein endothelial cells from gestational diabetic
pregnancies are consistent with some studies reporting an increase in basal NO synthesis in
diabetes (Corbett et al., 1992), but at variance with others showing an impaired synthesis
of NO in diabetes (see Poston and Taylor, 1995). Direct extrapolation of findings in cultured
endothelial cells to types 1 or 2 diabetic patients should be treated with caution, though
many outstanding questions relating to control of NO biosynthesis in the diabetic state can
now be addressed at a cellular level. Interestingly, NO synthesis and NO-mediated relaxation
of isolated resistance arteries studied in vitro do not appear to be impaired (McNally et al.,
1994; Hicks et al., 1997) in type 1 or 2 diabetic patients, whereas studies in vivo have
pointed to impaired endothelium-dependent relaxation in diabetic patients (see section
above). The finding that human insulin (and glucose) stimulate NO synthesis in cultured
fetal endothelial cells is consistent with the hypothesis that insulin activates the L-arginine/
NO vasodilator pathway in human skeletal muscle vasculature (see Baron, 1994; Scherrer
et al., 1994; Steinberg et al., 1994).
Strategies to Improve Endothelial Function in Diabetes

Interventions that may improve endothelial function in diabetic patients include fish oil
(McVeigh et al., 1993, 1994), water soluble antioxidants in type 2 diabetes and lipid
lowering therapy in type 1 diabetes (Mullen et al. 1998). Many other interventions hold
therapeutic promise. Lifestyle changes to reduce insulin resistance and improve glycaemic
control and dyslipidaemia through diet and exercise may be especially important.
Angiotensin converting enzyme (ACE) inhibitors might improve endothelial function by
preventing the generation of superoxide anions by angiotensin II and indirectly by
increasing bradykinin-stimulated NO synthesis. In patients with coronary artery disease
the ACE inhibitor quinapril reverses endothelial dysfunction in coronary vessels
independent of alterations in plasma lipid profiles and blood pressure (Mancini et al.,
1996), but whether such an effect occurs in patients with diabetes is unknown. Angiotensin
II (AT1) receptor antagonists may reduce O2- production by blocking effects of angiotensin
II on NADH/NADPH oxidase (Rajagopalan et al., 1996). Moreover, statins improve
endothelial function in coronary and peripheral vessels of non-diabetic patients with
hypercholesterolaemia(Stroes et al., 1995; Treasure et al., 1995; O’Driscoll et al., 1997).
Promising agents for trials in diabetes include fibrates (e.g. ciprofibrate) and statins (e.g.
atorvastatin), which have powerful effects on serum triglyceride and LDL-cholesterol
concentrations and influence LDL particle size. Thiazolidonediones could potentially
reverse endothelial dysfunction by enhancing insulin sensitivity (Nolan et al., 1994) and
decreasing lipoprotein oxidation (Noguchi et al., 1996), providing the hepatotoxicity of
troglitazone does not prove a general feature of this class of drug. Clinical trials to
determine effects of many of these interventions on endothelial function are in progress.
The link between endothelial dysfunction and insulin resistance suggests that interventions
which improve endothelial function could also improve insulin sensitivity in type 2
diabetes.
ACKNOWLEDGEMENTS
We gratefully acknowledge the support of the Wellcome Trust, British Heart Foundation
and Ministry of Agriculture, Fisheries and Food.
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Flow-mediated dilatation of conduit arteries is dependent on intact endothelial function, and is largely (but not
exclusively) mediated by endothelium-derived nitric oxide (NO). As arterial diameter can be measured accurately
in humans in vivo by either quantitative angiography (invasive) or high resolution external vascular ultrasound
(non-invasive), human studies of flow-mediated dilatation have recently been described, and have provided insights
into the relationships between cardiovascular risk factors and endothelial physiology. In the last 5 years, impaired
activity of endothelium-derived NO has been demonstrated in children and young adults with hypercholesterolemia,
diabetes mellitus or with a history of cigarette smoking. Non-invasive assessment of flow-mediated dilatation in
particular has facilitated study of arterial physiology in younger presymptomatic subjects, relatively early in the
atherogenic process. This methodology has also permitted serial study of endothelium-dependent dilatation, to
permit clinical trials of agents which may reverse endothelial dysfunction in high risk subjects. Amongst other
interventions, oral L-arginine (the substrate of NO), vitamin C and estrogen replacement therapy have shown
promise as agents that might improve arterial endothelium-dependent dilatation. As endothelium-derived NO
may play a role, not only in vasodilatation, but also in regulating monocyte adhesion, platelet aggregation and
smooth muscle proliferation, such agents may have potentially important effects on the development of
atherosclerosis.
Key words: Endothelial function; non-invasive detection; high resolution ultrasound; risk factors.
10 Flow-Mediated Dilatation and Cardiovascular Risk
David S.Celermajer1 and John E.Deanfield2

1
Department of Cardiology, Royal Prince Alfred Hospital, Camperdown, Sydney NSW 2050,
Australia
2
Cardio-Thoracic Unit, Great Ormond Street Hospital for Children, London, WC1 3JH, UK
FLOW-MEDIATED DILATATION
Increasing blood flow through large arteries produces an increase in vessel diameter, both
in animals (Hintze and Vatner, 1984) and in man (Anderson and Mark, 1989, Sinoway et
al., 1989, Laurent et al., 1990). This phenomenon of flow-mediated dilatation (FMD) is
now known to be endothelium-dependent, as a normal arterial response is critically dependent
on the presence of a functionally intact endothelium (Smiesko et al., 1985, Pohl et al.,
1986). The fact that FMD is endothelium-dependent forms the basis for in vivo testing of
endothelial function in human subjects.
Control of vessel diameter by changes in blood flow was first proposed in 1933
(Schretzenmayr, 1933), who observed dilatation of the canine femoral artery in response to
increases in femoral blood flow. Originally the mechanism of this vasodilatation was thought
to be due to a retrograde wave of dilatation starting in the arterioles and spreading to the
large proximal arteries; so called “ascending dilatation” (Macho et al., 1981). This was
soon disproved, however, after experiments in which cutting the femoral artery distal to an
arteriovenous fistula failed to abolish FMD (Ingebrigtsen and Leraand, 1970; Lie et al.,
1970) (Figure 10.1).
It was hypothesized therefore that a local mechanism controls arterial responses to flow.
Endothelial cells are ideally situated to “transduce” the signal of increased flow/shear stress
into a vessel wall response via modulating the production and release of vasoactive
substances. Endothelial cells in culture are known to react to changes in mechanical forces
by altering metabolism (Franke et al., 1984; Van Grondelle et al., 1984).
Smiesko et al. (1985) and Pohl et al. (1986) have shown that FMD occurs in normal
arteries and is abolished completely by techniques used to injure the endothelium (mechanical
or pharmacological). Furthermore Rubanyi et al. (1986) showed that FMD is related to the
release of an endothelium-derived relaxing factor, and Gruetter et al. (1981) demonstrated
abolition of FMD by the concomitant administration of the EDRF inhibitor, methylene
blue. Recently, Joannides et al. (1995) have shown that FMD in human arteries is mediated
predominantly by NO release, as it can be largely attenuated by coadministration of L-
NMMA, an inhibitor of NO synthesis.
There is some temporal delay between the increase in flow in an artery and the subsequent
flow-associated dilatation (Coretti et al., 1995). This raises the question as to whether
increased flow itself mediates the dilatation, or sets up another mechanism of sustained
physiologic change which in turn mediates the vasodilation (Bhagat et al., 1997).
Both absolute flow increase and pulsatility are powerful stimuli for endothelium-
dependent, flow-associated dilatation. The mechanism whereby endothelial cells sense an
191
192 D.S.Celermajer and J.E.Deanfield
Figure 10.1 Continuous vessel diameter showing brachial artery dilatation after inflation and release of a pneumatic
tourniquet on the forearm (flow mediated endothelial dependent dilatation) followed by dilatation to sublingual
nitroglycerin (endothelial independent dilatation). High resolution ultrasound has been used to study conduit
artery physiology (see text) non-invasively in children from 5 years of age. (Courtesy of Dr M.J.Mullen, Great
Ormond Street Hospital for Children, London, UK)
increase in shear stress is not well characterized, but is probably mediated via activation of
a K+ channel in the endothelial cell membrane (Rubanyi et al., 1990). Vascular relaxation
may also occur by direct transmission of endothelial cell hyperpolarization to the underlying
smooth muscle via gap junctions (Olesen et al., 1988).
TESTING ENDOTHELIAL FUNCTION IN HUMANS
To date, most studies of endothelial function in humans have relied on measurement of

endothelium-related changes in vasomotor function (even though this is only one of many
aspects of endothelial physiology). In 1986, Ludmer et al. reported the first studies of
coronary endothelium-dependent dilatation and constriction in adults with chest pain
syndromes; these studies relied on invasive instrumentation of the coronary arteries,
infusion of vasoactive compounds such as acetylcholine and serial measurement of arterial
diameter by quantitative angiography. This and a number of subsequent studies of coronary
vasomotion detected impaired endothelium-dependent dilatation in subjects with
established coronary stenoses and/or with risk factors for atherosclerosis (Nabel et al.,
Flow-Mediated Dilatation and Cardiovascular Risk 193
1990; Zeiher et al., 1991; Yeung et al., 1991). In addition to using pharmacological agents
such as acetylcholine or serotonin to stimulate endothelium-derived vasoactive mediators,
some of these studies measured flow-mediated vasodilatation (induced by exercise, cardiac
pacing or distal papaverine infusion—for example, see Gordon et al., 1989). These early
studies of coronary FMD, however, were limited to relatively small numbers of already
symptomatic subjects.
Invasive studies of coronary vasomotion are clearly inappropriate for studying
presymptomatic children and young adults, early in the natural history of the atherogenic
process. For this reason, non-invasive testing of peripheral conduit arteries was developed.
In 1992, we described a non-invasive method for measuring brachial and femoral artery
FMD in humans (Celermajer et al., 1992). This method had the same principles as the
coronary endothelial studies described above, however the endothelial stimulus was provided
by increased flow rather than by infused agents, and vessel diameter was measured by
ultrasound, rather than by angiography.
THE NON-INVASIVE TECHNIQUE FOR MEASURING FMD
Serial measurements of the diameter of a target artery can be obtained using high resolution
B-mode images, with commercially available 7–10 MHz linear array transducers and
standard ultrasound mainframes. Only relatively superficial arteries can be well visualized
using these transducers (such as the brachial, femoral or carotid arteries, but not the coronary
arteries). Arterial diameter is measured at rest and during a condition of increased flow (for
example, produced by distal cuff occlusion and release), resulting in flow-mediated,
endothelium-dependent dilatation. The arterial diameter is usually also measured after
sublingual nitroglycerin, which is an endothelium-independent dilator.
In practice, the subject lies at rest for at least 10 minutes before the first ultrasound
scan (baseline). The target artery (either the superficial femoral artery just distal to the
bifurcation of the common femoral, or the brachial artery 2–15 centimetres above the
elbow) is scanned in longitudinal section. The center of the artery is identified when
the clearest picture of the anterior and posterior intimal layers is obtained. The transmit
(focus) zone is set to the depth of the near wall, in view of the greater difficulty of
evaluating the near compared to far wall “m” line (the interface between media and
adventitia) (Pignoli et al., 1986, Nolsoe et al., 1990). Our usual settings are a transmit
power of–9dB, log compression 40dB and overall gain 3 dB. Individual depth and gain
settings, however, are set to optimize of the lumen/arterial wall interface. Images are
magnified using a resolution box function (leading to a television line width of
approximately .065 mm), and machine oper ating parameters are not changed during
any study. The processing curves are chosen to enhance the vessel wall/lumen interface
(high contrast, crisp borders).
When a satisfactory transducer position is found, the skin is marked, and the limb remains
in the same position throughout the study. Care is taken to apply the transducer without
undue pressure. A resting scan is recorded and increased flow is then induced by inflation
of a pneumatic tourniquet to a pressure of 250–300 mmHg for 4.5 minutes, followed by
release. A second scan is taken for 30 seconds before and 90 seconds after cuff deflation, to
allow measurement of FMD. Thereafter 10 minutes is allowed for vessel recovery, after
which a further resting scan is taken. Sublingual nitroglycerin spray (400 mcg) is then
Figure 10.2 Experiments using a ‘phantom’ of 10 cylinders (arteries) demonstrating accuracy of ultrasound
measurements of arterial diameter measurements of 0.1 mm and 0.2 mm. (From Sorensen, K.E., Celermajer,
D.S., Spiegelhalter, D.J., Georgakopoulos, D., Robinson, J., Thomas, O., Deanfield, J.E. Non-invasive measurement
of human endothelium dependent arterial responses: accuracy and reproducibility. Br. Heart J., 1995;74:247–53.
Reproduced with permission from the publishers and authors.)
administered, and 3–4 minutes later the last scan is performed. The electrocardiogram is
monitored continuously.
Data analysis is undertaken off-line, using recorded images. Vessel diameter is measured
by 2 observers, who are unaware of the condition of the subject and the stage of the
experiment. The arterial diameter is measured at a fixed distance from an anatomical marker,
such as a bifurcation, using ultrasonic calipers. Measurements are taken from the anterior
to the posterior “m” line (the interface between the median and adventitia) at end-diastole,
incident with R-wave on electrocardiogram (ECG). For the reactive hyperemia scan, diameter
measurements are taken 45–60 seconds after cuff deflation. Four cardiac cycles are analysed
for each scan and the measurements averaged. The vessel diameter is measured in each of
the scans (that is, at rest, after reactive hyperemia, after a further 10 minutes rest and 3–4
minutes after nitroglycerin), and are then expressed as a percentage relative to the first
control scan (100%).
The best arteries to study using this technique are the brachial and superficial femoral,
as they are large and superficial systemic arteries in which reactive hyperemia can be induced
easily. As atherosclerosis is a diffuse process, which initially often follows a parallel course
in the medium-sized muscular arteries, our preliminary studies were carried out on the
superficial femoral artery. When we found an inverse relationship between flow-mediated
dilatation and resting vessel size in normal subjects (Celermajer et al., 1992), we confined
our study to arteries with diameter ≤ 6.0 mm; that is, superficial femoral arteries in children
and brachial arteries in adults.
Since this initial description, many modifications have been made by different groups;
all, however, rely on the accurate non-invasive measurement of flow-mediated arterial
dilatation. Different devices have been used to ensure stability of the ultrasound images
(e.g. stereotactic clamps) and various techniques have been reported for diameter
measurement (eg B-mode, A-mode “wall tracking”). In addition, semi-automated and
computerized methods for on-line or off-line diameter measurement have been described
(for example, Leeson et al., 1997). These modifications may have, in part, resulted in the
different ranges of “normal” FMD values reported by different investigators; certain groups
have reported higher (Coretti et al., 1995) or lower (Joannides et al., 1995) values for FMD
in healthy subjects than the control values for FMD reported by our groups (Adams et al.,
1996). This suggests that within laboratory comparisons of FMD values for subject groups
will be much easier than comparison of FMD values obtained by different groups, using
various modifications of the original methodology.
ACCURACY, REPRODUCIBILITY AND LIMITATIONS OF

MEASURING FMD
Accurate assessment of FMD depends on reliable detection of submillimeter differences

between diameter measurements. Using arterial phantoms, 7MHz ultrasound and B-mode
images, Sorensen et al. (1995) have demonstrated that diameter differences of 0.1–0.2
mm can be dectected reliably, in vessels measuring 2–5 mm in diameter. In this same
study, FMD was relatively reproducible in 40 subjects studied on 4 occasions each
(coefficient of variation <2%). Since this time, however, Hashimoto et al. (1996) have
demonstrated important cyclical variations of FMD in menstruating females, and Vogel
et al. (1997) have found significant changes in FMD after a high-fat meal; for these
reasons, time of cycle and post-prandial status need to be carefully controlled in any
studies of serial changes in FMD.
Limitations of measuring FMD include the difficulties in maintaining stable transducer
position (requiring a skilled sonographer and cooperative subject), the time consuming
process of image analysis, and the lack of access to coronary arteries with external ultrasound.
Furthermore, there is a wide range of “normal” values for FMD, even in healthy young
subjects without identifiable vascular risk factors (Adams et al., 1996). For these reasons,
we do not believe that FMD is a useful screening tool for endothelial dysfunction; it does,
however, remain a very useful test for studies of endothelial physiology in “at risk”
populations and for examining reversibility of early arterial damage in certain groups of
subjects.
Although brachial artery study per se is not clinically relevant, as brachial atherosclerosis
is an extremely infrequent health problem, it appears that brachial FMD is well correlated
with both coronary endothelial responses (Anderson et al., 1995) and with the extent and
severity of coronary atherosclerosis (Neunteufl et al., 1997). For these reasons, study of
peripheral artery FMD represents a useful surrogate for coronary abnormalities, particularly
in younger subjects (Sorensen et al., 1997).
THE EFFECT OF RISK FACTORS ON FMD
Impairment of conduit artery FMD is an early systemic event in children and adults with
risk factors for atherosclerosis. The effect of most of the traditional risk factors on FMD
has been well studied in the 1990’s, illustrating impaired endothelial release of NO early in
life in high risk subjects, and that most traditional vascular risk factors demonstrate a “dose-
response” relationship with impaired FMD.
Active cigarette smoking is associated with reduced FMD in a dose-dependent manner,
with FMD being almost completely absent in smokers with >20 pack year smoking history
(Celermajer et al., 1993) (Figure 10.3). Although this is potentially reversible, the degree
of reversibility of smoke-related endothelial damage may be minor. Even heavy exposure
to environmental tobacco smoke (“passive” smoking) has been associated with a dose-
related impairment of FMD, in otherwise healthy teenagers and young adults (Celermajer
et al., 1996).
Familial hypercholesterolemia (FH) is associated with profound impairment of FMD,
even in the first decade of life (Sorensen et al., 1994) (Figure 10.4). The degree of impairment
correlates with both LDL and Lp(a) levels, in young FH subjects. In non-FH subjects,
however, with total cholesterol levels from 3–6.5 mmol/L, cholesterol level is only weakly
(inversely) related to FMD (Celermajer et al., 1994b). Less information is available about
the effects of LDL, HDL, Lp(a) or triglyceride levels on FMD, in otherwise healthy, non-
FH subjects. Recently, however, Lundman et al. (1997) have demonstrated that acute infusion
of triglycerides is associated with impaired endothelium-independent vasodilatation in
healthy young adults.
The relationship between blood pressure and FMD is not well studied. In a group of
young adult survivors of aortic coarctation repair, exercise-related hypertension was related
to an impaired nitroglycerin response, but not to FMD (Gardiner et al., 1994). Studies of
FMD in essential hypertensive subjects, however, have not yet been reported, possibly
because of the confounding influence of anti-hypertensive agents on these tests of arterial
physiology.
Data on FMD in insulin-dependent diabetics have been conflicting, with one group
showing no change compared with controls (Lambert et al., 1996), and another larger study
showing significant impairment of both FMD and nitroglycerin responses (Clarkson et al.,
1996b). In this latter study, the degree of FMD impairment correlated with the duration of
disease and with the LDL cholesterol level.
A positive family history of premature coronary disease is associated with a significant
decrease in arterial FMD in otherwise health young subjects (Clarkson et al., 1997). This
is particularly marked if the index relative with premature coronary disease had no
identifiable vascular risk factors themselves, consistent with a heritable component in
the FMD response.
Although males have lower FMD values than females, this is accounted for completely
by their larger vessel size (Celermajer et al., 1992). In young subjects (<age 40 years),
there appears to be no gender difference in vessel size adjusted FMD; with aging, however,
there is a progressive decline in FMD, and this occurs earlier in males than in females
Figure 10.3 Smoking and brachial artery flow mediated dilatation. Impaired endothelial dependent dilatation is
present in association with cigarette smoking in 80 young asymptomatic subjects (top) in proportion to extent and
duration of smoking (bottom). (From Celermajer, O.S., Sorensen, K.E., Georgakopoulos, D., Bull, C., Thomas,
O., Robinson, J., Deanfield, J.E. Cigarette smoking is associated with dose-related and potentially reversible
impairment of endothelium-dependent dilation in healthy young adults. Circulation, 1993;88:2149–55. Reproduced
with permission from the publishers and authors).
Figure 10.4 Cholesterol and femoral artery flow mediated dilatation. Impairment of endothelial dependent dilatation
is A.L.ready present by the first and second decades of life (30 children age 7 to 17 years) in association with
familial hypercholesterolaemia (28 heterozygotes: 2 homozygotes in black). (From Sorensen, K.E., Celermajer,
O.S., Georgakopoulos, D., Hatcher, G., Betteridge, D.J., Deanfield, J.E.Impairment of endothelium-dependent
dilation is an E.A.rly event in children with familial hypercholesterolemia and is related to the lipoprotein (a)
level. J. Clin. Invest., 1994;93:50–55. Reproduced with permission from the publishers and authors).
(Celermajer et al., 1994a). This is consistent with the data of Hashimoto et al. (1996),
who showed increased FMD during the follicular phase of normal menses in young
women, suggesting an association between physiologic levels of estrogens and improved
arterial FMD.
The effect of relatively newly identified risk factors for premature atherosclerosis has
also been examined. The ACE “DD” genotype, which has been linked to a high risk of
myocardial infarction (Cambien et al., 1992), is not associated with impaired FMD
(Celermajer et al., 1994c). Hyperhomocysteinemia, however, is a risk factor for both
premature arterial disease and for impaired FMD, in both Caucasian (Tawakol et al., 1997)
and Chinese subjects (Woo et al., 1997a). As most endothelial function studies have been
carried out in Caucasian populations, it will be important to establish whether risk factors
interact with endothelial physiology in different ways in other ethnic groups. Some
preliminary data, for example, suggest that aging and smoking may have less effect on
FMD in Chinese compared to Caucasian subjects (Woo et al., 1997b, 1997c).
Most studies have examined the effects of single risk factors on FMD, in highly selected
populations. It appears, however, that in an unselected population of healthy subjects, that
Figure 10.5 One month of dietary supplementation with L-arginine improves brachial artery endothelial function
in young subjects with moderate hypercholesterolaemia studied in a double blind crossover trial. (From Clarkson,
P., Adams, M.R., Powe, A.J., Donald, A.E., McCredie, R., Robinson, J., McCarthy, S.N., Keech, A., Celermajer,
D.S., Deanfield, J.E. Oral L-arginine improves endothelium-dependent dilation in hypercholesterolemic young
adults. J. Clin. Invest., 1996;97:1989–94. Reproduced with permission from the publishers and authors).
traditional risk factors may interact with each other, to increase the risk of impaired arterial
FMD (Celermajer et al., 1994b). Similar observations have been made in the coronary
circulation of older symptomatic subjects (Vita et al., 1990).
REVERSIBILITY OF IMPAIRED FMD
Identification of subjects with impaired FMD (and therefore reduced endothelial NO release)
raises the question of reversibility of this abnormality, by risk factor modification, drug
therapy or other novel methods. The availability of a non-invasive technique for studying
FMD, which can be performed serially, facilitates the study of such potentially FMD-
enhancing strategies. Based on reproducibility data, power function analyses have been
performed (Sorensen, 1995), to allow accurate calculation of sample sizes required for
proposed interventional trials.
To date, 3 interventions have been shown to improve FMD with short-term oral
administration. L-arginine is the substrate for endothelial nitric oxide production, and
Clarkson et al. (1996a) have demonstrated that oral L-arginine (7 g three times daily, given
for 4 weeks) improves FMD in hypercholesterolemic young adults with endothelial
dysfunction (Figure 10.5). Similar beneficial effects on endothelial NO release in
hypercholesterolemic rabbits have been associated with a marked decrease in aortic
atherosclerosis (Cooke et al., 1992).
Similarly Levine et al. (1996) have assessed the effect of vitamin C on arterial FMD in
adults with established coronary disease, and shown acute improvement in FMD after a
single oral 2g dose. The mechanism of this effect is uncertain, but may be due to ascorbate-
related scavenging of oxygen-derived free radicals, with consequently increased
bioavailability of endothelium-derived NO. It is not yet known, however, whether the
beneficial effects of L-arginine or vitamin C will be sustained with long-term administration,
or whether this will be associated with a reduction in clinically relevant endpoints.
Finally, estrogen replacement therapy (ERT) has been shown to improve FMD in post-
menopausal women (Lieberman et al., 1994); this may explain some of the apparent
cardioprotective effects of such therapy in older females. As most older women require
progesterone co-supplementation with ERT, however, to protect the uterus, prospective data
on the effect of combined therapy on endothelial function are awaited; preliminary reports
are reassuring (McCrohon et al., 1996).
In addition to the above, trials on the effects of cholesterol lowering therapy, ACE
inhibition and calcium channel blockade on arterial FMD are underway in certain high risk
asymptomatic subjects; such studies may provide insights into the possible vascular protective
effects of these agents, early in the natural history of atherosclerosis.
CONCLUSIONS
Non-invasive measurement of flow-mediated, endothelium-dependent arterial dilatation has

provided important insights into the relationships between vascular risk factors and this
important aspect of endothelial physiology, in human subjects. The availability of an
ultrasound-based method for studying FMD has facilitated serial studies which have assessed
strategies for improving endothelial NO release, with promising early results. Future research
will focus on standardization and automation of FMD measurements, and on further trials
to determine the sustainability and clinical relevance of therapies which improve FMD, in
children and young adults at risk of atherosclerosis.
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Prostaglandins, nitric oxide (NO) and endothelins are endothelium-derived mediators that have important
physiological roles in the regulation of the systemic and renal vasculature. Within the kidney NO and prostaglandins
have mainly vasodilator and natriuretic effects, whereas endothelin pathways predominantly increase vascular
tone and reduce sodium excretion. Moreover in human renal disease there is evidence that the activity of these
mediators is altered, and might contribute to systemic hypertension, reduced renal perfusion and glomerular
filtration, or inflammatiry renal disease. Consequently manipulation of these mediators, either singly or in concert,
might improve the treatment of renal disease and its complications.
Key words: Nitric oxide, endothelin, prostaglandins, kidney, human.

11 Endothelial Mediators and Renal Disease
Raymond J.MacAllister1 and William B.Haynes 2

1
Centre for Clinical Pharmacology, The Wolfson Institute for Biomédical Research,
University College London, 140 Tottenham Court Road, London W1P 9LN, UK
2
Department of Internal Medicine, University of Iowa, Iowa City, IA 52241, USA
INTRODUCTION
As in other vascular beds, the endothelium of the renal vasculature synthesises mediators
such as NO, prostaglandins and endothelin with potential to modulate blood vessel function.
However, the endothelium is not the only cell type that synthesises these autacoids, and
many renal parenchymal cells synthesise and respond to these mediators. This makes it
difficult to be certain of the precise contribution of the endothelium to effects mediated by
these agents. Moreover, the kidney is also responsible for the excretion of substances that
might alter endothelial function, and as a result renal dysfunction could contribute to
widespread endothelial abnormalities. In this chapter, we will review the role of endothelial
mediators in the regulation of renal function with emphasis on human physiology and
pathophysiology.
OVERVIEW OF THE PHYSIOLOGICAL AND

PATHOPHYSIOLOGICAL ROLES OF ENDOTHELIUM-DERIVED
MEDIATORS IN THE SYSTEMIC VASCULATURE
The vascular endothelium generates vasoactive mediators including nitric oxide (NO),
prostanoids and endothelins (Figure 11.1). Endothelial cyclooxygenase-1 (COX-1) generates
prostanoids from arachidonic acid, some of which cause smooth muscle relaxation
(prostacyclin, prostaglandin E) by stimulating adenylate cyclase through G-protein coupled
receptors (Coleman et al. 1994). The vasoconstrictor prostanoid thromboxane acts on its
receptors to mobilise intracellular calcium causing smooth muscle contraction. Endogenous
prostanoids do not appear to modulate systemic vascular tone in healthy humans, because
inhibition of COX-1 by nonsteroidal anti-inflammatory drugs (NSAIDs) has little effect on
systemic vascular resistance and blood pressure (Taddei et al., 1993; Pope et al., 1998).
However, in vascular disease, prostaglandins might be more important in the regulation of
vascular tone (Taddei et al., 1997; Johnson, 1997). A pathophysiological role for constrictor
and pro-thrombotic prostanoids is also supported by the therapeutic effect of aspirin in
patients with vascular disease (Thiemermann et al., 1993).
Nitric oxide (NO) is synthesised by NO synthase in the vascular endothelium from the
amino acid L-arginine. NO diffuses to the vascular smooth muscle where many of its effects
are mediated by stimulation of soluble guanylate cyclase (sGC) to generate cyclic GMP
(Moncada et al., 1993). In contrast to COX inhibition, inhibition of NO synthesis in healthy
subjects increases vascular tone and blood pressure, indicating that under physiological
205
206 R.J.MacAllister and W.B.Haynes
Figure 11.1 Simplified schematic of the NO, endothelin and prostaglandin pathways. In the kidney many cell
types synthesise and respond to NO, endothelin and prostaglandins. NO is generated from L-arginine by the
action of NO synthase enzymes (NOS) and many of its effects are a consequence of stimulation of soluble guanylate
cyclase (sGC) to generate cyclic GMP (cGMP). Endothelin (ET) is produced from pro-endothelin (pro-ET) by
endothelin converting enzyme (ECE). Endothelin stimulates cell surface receptors (ET-R) which mediate smooth
muscle contraction by activation of phospholipase C (PLC). Cyclooxygenase enzymes (COX) metabolise
arachidonic acid to a variety of prostaglandins (Pg) and these activate cell surface receptors causing constriction
(mediated by PLC) or dilatation due to stimulation of adenylate cyclase (AC) to increase intracellular cyclic AMP
(cAMP). In addition to these pathways, NO, endothelin and prostaglandins have been implicated in the modulation
of the function of ion channels and ion pumps in the target cell membrane, and these actions could also contribute
to their biological effects.
conditions, there is background NO-mediated dilatation of the arterial vasculature of humans

which is a determinant of blood pressure (Moncada et al., 1993, see chapter 1). In addition
to effects on vascular tone, NO decreases proliferation of underlying vascular smooth muscle,
maintains endothelial cell impermeability, inhibits platelet aggregation and adhesion and
inhibits activation and adhesion of leucocytes to endothelium (Kanwar et al., 1995; Radomski
et al., 1993). These additional effects of NO might protect the vasculature from the
development of atheroma and its complications (Cooke et al., 1997). In patients at risk of
developing atheroma, and in the presence of established disease, there is evidence for reduced
NO-mediated effects. Whether NO-replacement therapy is anti-atherogenic remains to be
determined.
The endothelins are a family of endothelium-derived peptides that possess
characteristically sustained vasoconstrictor properties (Yanagisawa et al., 1988). The three
endothelin isopeptides are generated through cleavage of precursor big endothelin molecules
to the mature peptides by endothelin converting enzymes (Inoue et al., 1989). Endothelin-
1 appears to be the predominant member of the family generated by vascular endothelial
cells (Howard et al., 1992), so our discussion focuses on this isopeptide. Endothelin exerts
Endothelial Mediators and Renal Disease 207
its actions by binding to at least two receptors, the ETA and ETB subtypes. In addition to its
direct vascular effects, endothelin-1 has inotropic and mitogenic properties, influences salt
and water homeostasis, alters central and peripheral sympathetic activity, and stimulates
the renin-angiotensin-aldosterone system (Haynes et al., 1998). Studies with endothelin
receptor antagonists have indicated that endothelin-1 has complex opposing vascular effects
mediated through vascular smooth muscle and endothelial ETA and ETB receptors.
Endogenous generation of endothelin-1 appears to contribute to maintenance of basal
vascular tone and blood pressure through activation of vascular smooth muscle ETA receptors
(Haynes et al., 1994; Haynes et al., 1996). At the same time, endogenous endothelin-1 acts
through endothelial ETB receptors to tonically stimulate nitric oxide formation and oppose
vasoconstriction (Verhaar et al., 1998).
Endothelium-derived hyperpolarising factor has also been implicated in the dilator
function of the endothelium (see chapter 4). This substance has yet to be identified but
appears to contribute to endothelium-dependent dilatation of the renal resistance vasculature
in animals (Vargas et al., 1994). Its role in humans has yet to be investigated and will not be
discussed further in this chapter.
DISTRIBUTION AND BIOCHEMISTRY OF ENDOTHELIAL

MEDIATORS IN THE HUMAN KIDNEY
Biochemical and molecular studies have been used to confirm that the human kidney has
the capacity to synthesise and respond to NO, prostaglandins and endothelins.
Cyclo-Oxygenase Expression and Activity

Immunohistochemistry and in situ hybridisation has identified constitutive expression
of COX-1 and COX-2 in human collecting duct, endothelial and smooth muscle cells
in afferent and efferent arterioles, and in the medullary vessels (Kömhoff et al., 1997).
COX-1 predominates in the medulla, COX-2 in the cortex. In animals there is constitutive
expression of COX-2 in the macula densa, which is increased by sodium-depletion. In
contrast, there is no expression of COX-2 in the human macula densa and it remains to
be determined whether human COX-2 expression can be induced. There is regional
variation of prostaglandin synthesis in the human kidney; renal vessels synthesise
prostacyclin and PGE2, the glomeruli synthesise prostacyclin, the medulla mainly PGE2,
and in the renal pelvis and ureters PGE2 and thromboxane predominate (Coleman et
al., 1990). There is limited data on the distribution of prostaglandin receptors in the
human kidney, but PGE binding sites occur at highest density in the renal medulla
(Eriksson et al., 1990).
NOS Distribution And Activity

Animal models suggest that all three isoforms of NOS are constitutively expressed in the
kidney (Bachman et al., 1995). In humans, there is evidence for expression of endothelial
NOS in the renal cortex (afferent and efferent arterioles), and medullary vessels (including
veins and vasa recta) (Bachman et al., 1995). nNOS has been detected by
immunohistochemistry in the macula densa, although staining appears to be less dense
than in animals. These areas also exhibit NADH diaphorase staining, which is consistent
with NOS activity. There is NOS activity in the renal pelvis (Iversen et al., 1995), and
immunohistochemistry has identified nNOS positive neurones in the human uterovesical
junction (Goessl et al., 1995). Although human proximal tubular cells in culture synthesise
NO (McLay et al., 1994; McLay et al., 1995) and mRNA for iNOS has been detected in
these cells, (McLay et al., 1994), the mRNA and protein for inducible NOS has only been
detected in renal biopsy specimens from patients with glomerulonephritis, where iNOS is
localised to immune cells and not the renal parenchyma (Kashem et al., 1996).
Endothelin And Endothelin Receptor Distribution

Although mRNA for all three endothelin isoforms can be detected in the medulla, cortex
and vasculature of human kidney, only big endothelin-1 can be detected by immunocy-
tochemistry (Karet et al., 1996). In human renal cortex, gene expression for
preproendothelin-1 is particularly evident in the endothelial layer of arcuate and interlobular
arteries and veins and in afferent arterioles (Pupilli et al., 1994). In renal medulla,
preproendothelin-1 is highly expressed in vasa recta bundles and capillaries, and collecting
ducts (Pupilli et al., 1994). Big endothelin-1 is cleaved to mature endothelin-1 by both
neutral endopeptidase and endothelin converting enzyme in human kidney (Russell et al.,
1998); the latter is highly expressed in renal arteries and arterioles, the loop of Henle and
collecting ducts (Pupilli et al., 1997). Human renal cortex expresses three times as much
mRNA for ETA as the ETB receptor (Karet et al., 1994). In contrast, the ETB receptor is
preferentially expressed in human renal medulla (Karet et al., 1994).
PHYSIOLOGICAL ROLES OF ENDOTHELIAL MEDIATORS IN THE

HUMAN KIDNEY
Regulation of Vascular Tone

In humans there is functionally active background production of NO, prostaglandins and
endothelin-1 in the kidney, which contributes to the regulation of resistance vessel tone and
renal blood flow under basal or stimulated conditions. It is assumed that the endothelium is
the source of these mediators, but this might not be the case.
Arachidonic acid (the precursor of prostaglandins) has not been given systemically to
humans, but in some animal species it causes renal vasodilatation, that is blocked by NSAIDs,
suggesting metabolism of arachidonic acid to dilator prostaglandins (Dibona, 1998). In humans,
infusion of exogenous prostaglandins (PGE2, prostacyclin and PGA) causes renal vasodilatation
(Dibona, 1998). The role of endogenous prostaglandins has been determined in humans by
inhibition of COX isoenzymes using non-steroidal antiinflammatory drugs (NSAIDs). In
healthy subjects in vivo, there is a small short term reduction in renal blood flow whereas
long-term use of NSAIDs has no significant effect on renal blood flow. It is not clear how the
kidney compensates during chronic COX inhibition (Dibona, 1998; Sedor et al., 1986). The
effects of NSAIDs are more marked following intra-vascular volume depletion when renal
blood flow falls by 10–20% (without changes in glomerular filtration rate) (Scharschmidt et
al., 1986). It is possible that hypovolaemia causes neurohormonal activation, and the resulting
renal effects of noradrenaline or angiotensin II are modulated by stimulation of dilator

prostaglandins. In addition, there is evidence in animal models that the effects of inhibition of
prostaglandin synthesis are also more marked in the presence of NO synthase inhibition
(Romero et al., 1992), suggesting that prostaglandin production might compensate for loss of
NO-mediated dilatation. Whether this is also true in humans remains to be determined.
There is compelling evidence in humans that NO modulates vascular tone in the renal
bed; human isolated vessels exhibit endothelium-dependent relaxation to agonists such as
acerylcholine or NO donors in vitro (Lüscher et al., 1987) and in vivo (Elkayam et al,
1996). Moreover, inhibition of NO synthase reduces renal blood flow and glomerular
filtration rate in vivo (Bech et al., 1996; Wolzt et al., 1997), suggesting that endogenous
NO exerts basal vasodilatation of the resistance vasculature of the kidney.
Endothelin-1 contracts afferent and efferent arterioles equally in vitro (Edwards et al.,
1990), and thus reduces both renal plasma flow and glomerular filtration rate in vivo in
humans (Sorensen et al., 1994; Rabelink et al., 1994). Endothelin-1-induced renal
vasoconstriction involves ETB receptors in the rat (Pollock et al., 1993) but mainly ETA
receptors in the dog and human (Brookes et al., 1994; Maguire et al., 1994). Renal
vasodilatation occurs following infusion of BQ-123 in dogs, suggesting that vascular
generation of endothelin-1 contributes to basal renal vascular tone (Pollock et al., 1993). In
humans, endothelin receptor antagonists decrease effective renal vascular resistance by
approximately 10%, without changing GFR (Webb et al., 1998, Ferro et al., 1997),
suggesting that the predominant effect of endogenously generated endothelin-1 is on the
efferent arteriole. These data support a physiological vasoconstrictor role for endogenous
endothelin-1 through activation of ETA receptors in human kidney.
Regulation of Sodium Excretion

In humans, the dilator prostaglandins PGE2 and prostacyclin cause natriuresis, which might
be due to their vasodilator action, or a direct effect on tubular sodium transport (Sedor et
al., 1986). NSAIDs reduce sodium excretion in healthy volunteers in the short term, but
this does not persist with chronic usage (Sedor et al., 1986). These effects of NSAIDs are
more marked in the presence of sodium depletion (Sedor et al., 1986). Whether sodium
retention explains the pressor effects of NSAIDs in certain patient groups remains to be
determined (Johnson, 1997). Other direct effects of prostaglandins on tubular function have
been suggested (such as inhibition of the activity of ADH) (Sedor et al., 1986), but the low
incidence of electrolyte disturbance caused by NSAIDs in humans suggests that these are
of minor functional importance in vivo.
In humans, inhibition of endogenous NO generation reduces sodium excretion (Bech et
al., 1996) but the mechanism of these effects have yet to be established. However, several
possibilities have been suggested by animal studies. NO could have direct effects on the
proximal tubule and the collecting duct to promote sodium and water loss. NO might also
promote natriuresis by increasing medullary blood flow and interstitial pressure. Local
inhibition of medullary synthesis of NO can lead to sodium retention and hypertension in
the absence of any direct effect on the systemic vasculature (Mattson et al., 1994).
Micropuncture studies of animal isolated nephrons have also implicated NO synthesised
by the macula densa in the modulation of the tubulo-glomerular feed-back (TGF). This
negative feedback mechanism regulates sodium excretion by eliciting afferent arteriolar
constriction if the sodium content of the distal tubular fluid is high, and this reduces the
delivery of sodium to the tubule. NO inhibits afferent arteriolar constriction, and inhibition
of NO could increase the sensitivity of this mechanism to reduce natriuresis (Wilcox et al.,
1992). The effects of NO on renin secretion are complex, with evidence from in vitro studies
of stimulation and inhibition of renin secretion by NO (Reid et al., 1995). However most
studies of intact animals suggest that NO stimulates renin secretion (Kurtz et al., 1998). In
humans inhibition of NO synthesis has no effect on plasma renin in sodium-replete
individuals (Dijkhorstoei et al., 1998), but blocks the rise in renin that occurs following
sodium depletion (Lee et al., 1996). It is therefore possible that the reninangiotensin system
provides feedback control of the natriuretic effects of NO.
Several lines of animal evidence suggest an important role for renal tubular endothelin-
1 in modulation of sodium and water excretion. Endothelin-1 blocks sodium and water
reabsorption by inhibiting tubular Na+/K+-ATPase activity in the proximal tubule and
collecting duct (Zeidel et al., 1989), and by inhibiting the effects of ADH on tubular osmotic
permeability in the collecting duct (Tomita et al., 1990; Oishi et al., 1991). In addition, the
cAMP response of rat collecting duct cells to ADH is potentiated in the presence of specific
endothelin-1 antisera, suggesting that endogenous production of endothelin-1 tonically
inhibits ADH responses (Kohan & Padilla, 1993). These tubular effects also occur with
ETB receptor agonists and are not blocked by the ETA receptor antagonist, BQ-123,
suggesting that they are mediated by ETB receptors (Kohan et al., 1993; Yukimura et al.,
1994). Involvement of ETB receptors is supported by the finding that ETB knockout mice
have hypertension secondary to renal sodium retention (Webb et al., 1998). Taken together,
these findings suggest that locally generated endothelin-1 plays a tonic physiological role
in regulation of salt and water transport in the terminal nephron, with increases in local
generation promoting natriuresis and diuresis via activation of ETB receptors. When
administered to humans endothelin-1 causes anti-natriuresis, presumably because ETA
receptor mediated vasoconstriction to exogenous endothelin-1 outweighs any diuretic or
natriuretic effect mediated by tubular ETB receptors (Rabelink et al., 1994; Sorensen et al.,
1994; Bijlsma et al., 1995). There are no data on the effects of selective ETB receptor
antagonism in humans.
In summary, inhibition of prostaglandin or NO synthesis, and blockade of endothelin
receptors has marked functional consequences on renal blood flow and tubular function.
The effects of prostaglandin synthesis inhibition are transitory, and this emphasises the
requirement for chronic studies of inhibition of NO and endothelin pathways in humans.
Moreover, studies of the effects of combined inhibition of endothelial mediators in humans
will increase understanding of the possible interaction of these systems within the kidney.
ENDOTHELIAL MEDIATORS IN ANIMAL MODELS OF RENAL

DISEASE
Methodological difficulties handicap detailed investigation of the role of endothelial

mediators in human kidney disease. Most of the human studies to date have concentrated
on endothelial function in the peripheral vasculature. However, the results of animal studies
have implicated abnormalities of the renal NO, prostaglandin and endothelin pathways.
Increased production of constrictor and dilator prostanoids has been reported in
experimental glomerulonephritis, acute renal failure, ureteric obstruction and renal transplant
rejection (Coleman et al., 1990), but it is unclear if they have a significant pathophysiological
role; neither thromboxane antagonists nor NSAIDs have been shown to consistently improve
renal function in these models (Stork et al., 1986). Indeed, infusion of dilator prostaglandins
has been shown to improve renal function in animal models possibly by increasing renal
blood flow or by modulating the immune response (Stork et al., 1986).
Reduced NO-mediated effects in the kidney have been documented in a variety of
experimental renal pathologies, including salt-sensitive hypertension, obstructive
nephropathy, subtotal nephrectomy, glomerular thrombosis in pregnancy and cyclosporin
nephropathy (Reyes et al., 1994; Wagner et al., 1995). Whether the systemic NO pathway
is abnormal in animal models of chronic renal failure is unclear, with evidence for normal
(Wagner et al., 1995), increased (Ye et al., 1997; Mendez et al., 1998; Aiello et al., 1997)
or reduced (Vaziri et al., 1998) activity of the pathway. In many of these conditions, renal
dysfunction is partially reversed by exogenous L-arginine, suggesting that absolute or relative
substrate deficiency has a pathogenic role (Reyes et al., 1994). In contrast, increased
production of NO secondary to iNOS induction, have been described in glomerular
inflammation (Cattell et al., 1993). High concentrations of NO in these conditions might
be cytotoxic, and in some studies inhibition of NO synthesis has a protective effect (Millar
et al., 1997).
Several factors should be considered in interpreting the results of studies of endothelin
in renal disease. First, the diverse actions of endogenous endothelin-1 on renal function
mean that any changes in endothelin-1 activity in models of renal disease must be interpreted
cautiously. Second, the kidney is an important site for clearance of endothelin-1 (Kohno et
al., 1989). Thus, increases in plasma concentrations in renal disease may be due to reduced
clearance of endothelin rather than increased generation. Third, urinary endothelin generation
is more likely to reflect renal tubule rather than vascular generation of endothelin. Given
that renal tubule generation of endothelin-1 appears to cause natriuresis, increases in urine
endothelin-1 generation may be beneficial. Fourth, interpretation of changes in sensitivity
to exogenous endothelin-1 may be confounded by the fact that endothelin receptor number
is downregulated by increased endothelin-1 concentrations (Hirata et al., 1988). Vascular
responses to exogenous endothelin-1 may also be altered by vascular remodeling. Fifth,
endothelin-1 appears to be primarily a locally acting paracrine substance rather than a
circulating endocrine hormone. Venous plasma endothelin-1 concentrations can only be
used as an approximation for endothelial synthesis of the peptide, though circulating
concentrations of big endothelin may more accurately reflect endothelin-1 generation
(Plumpton et al., 1995; Plumpton et al., 1996).
Increased circulating endothelin-1 concentrations and local endothelin-1 expression have
been reported in animal models of acute renal failure (secondary to hypoxia, septic shock
or radiocontrast media) (Shibouta et al., 1990; Marguiles et al., 1991; Firth & Ratcliffe,
1992; Krause et al., 1997). Renal vasoconstriction following ischaemia is substantially
ameliorated by administration of endothelin receptor antagonists (Gellai et al., 1994; Chan
et al., 1994; Clozel et al., 1993). Interestingly, in dogs, the ETA antagonist BQ-123 does not
prevent renal vasoconstriction after ischaemia (Stingo et al., 1993; Brooks et al., 1994),
even though ETA receptors appear to predominate in the canine renal vasculature (Karet &
Davenport 1994). Ischaemia-induced renal dysfunction in the dog is prevented by ETA/B
antagonists such as SB 209670 and L-754,142 (Brooks et al., 1994; Krause et al., 1997).
The vasoconstrictor effects of endothelin-1 are a plausible mechanism to underlie its
contribution to the pathophysiology of acute renal failure. However, the greater beneficial
effects of the combined ETA/B antagonist in a canine model suggests that endothelin-1 may
also act on non-vascular ETB receptors, including those situated on tubular epithelial cells.
There is also evidence that endothelin-1 may promote the marked neutrophil accumulation
observed in renal ischaemia-reperfiision injury through induction of the CD18 integrin
antigen (Espinosa et al., 1996).
Animal models of chronic renal failure are also associated with increased endothelin-
1 expression (Brooks et al., 1991; Benigni et al., 1991; Orisio et al., 1993; Nakamura et
al., 1995). Chronic administration of the ETA receptor antagonist FR139317 or of the
ETA/ B antagonist bosentan prevents the development of hypertension, glomerular damage
and renal insufficiency in a reduced renal mass model in rats (Benigni et al., 1993; Benigni
et al., 1996). It is not known whether ETA/B blockade is superior to ETA receptor blockade
in prevention of progressive renal damage. Endothelin-1 might also contribute to renal
damage in experimental hypertension, because treatment with endothelin antagonists
reduces the rate of deterioration of renal function (Okada et al., 1995; Karam et al.,
1996; Kohno et al., 1997).
ENDOTHELIAL MEDIATORS IN HUMAN DISEASE
In humans with renal disease there is evidence to suggest both increased and decreased
effects of endothelial mediators locally and systemically (Figure 11.2).
Prostaglandins
There is increased urinary excretion of dilator and constrictor prostaglandins in the urine of
patients with glomerulonephritis, suggesting increased renal production (Coleman et al.,
1990; Patrono et al., 1986). Functionally, the dilator effect of renal prostaglandins
predominates in disease states, because inhibition of COX with NSAIDs causes greater
reductions in renal blood flow and glomerular filtration rate in patients with nephrotic
syndrome (Vriesendorp et al., 1986) or chronic renal impairment (Patrono et al., 1986)
than in healthy controls. Although NSA IDs reduce proteinuria in nephrotic syndrome, this
is probably due to reduced renal blood flow and glomerular filtration, rather than an
immunomodulatory effect (Vriesendorp et al., 1986).
Abnormalities of the NO Pathway

Functional studies of the peripheral vasculature suggest that there is reduced endothelium-
dependent dilatation of veins (Hand et al., 1998) and conduit arteries (Kari et al., 1997) in
the systemic vasculature of patients with renal failure, consistent with reduced NO-mediated
effects. Whether the NO pathway in the kidney is abnormal in human renal disease remains
to be determined. The direct effects of L-arginine on the renal vasculature has been used to
assess endothelial function, based on the assumption that any effect of L-arginine is due to
stimulation of NO production (Gaston et al., 1995; Andres et al., 1997; Campo et al., 1996).
In healthy subjects, L-arginine increases renal blood flow, and glomerular filtration, effects
that are diminished in hypertensive subjects (Higashi et al., 1997). However, the large doses
used in most of these studies are likely to have non-specific effects on tissues. When given
to achieve similar concentrations, D-arginine (which is not a substrate for NO production)
also causes vasodilatation (Calver et al., 1991; MacAllister et al., 1995; Amodeo et al.,
Figure 11.2 Physiological and pathophysiological roles for endothelial mediators. Alteration in the activity of the
NO, prostaglandin or endothelin pathways has been documented in the kidney and the systemic vasculature of
patients with renal disease.
1996). Furthermore L-arginine has other NO-independent effects that could cause
vasodilatation (MacAllister et al., 1995; Higashi et al., 1995). Therefore, it is difficult to
draw clear conclusions about the NO pathway from clinical studies in which high doses of
L-arginine have been used.
Impaired renal function per se might result in widespread effects on systemic NO
pathways due to the accumulation of guanidino compounds that inhibit all three NOS
isoforms. These include analogues of L-arginine, such as L-NMMA and NGNGdimethyl-L-
arginine (ADMA) (Vallance et al., 1992). Plasma concentrations of ADMA are increased
in patients with chronic renal failure (Arese et al., 1995; MacAllister et al., 1996), and the
ratio of ADMA to L-arginine is increased approximately four-fold (MacAllister et al., 1996).
Therefore, it is possible that the increase in ADMA concentration could be responsible for
inhibition of NO synthesis in the uraemic state. ADMA might exert direct effects on systemic
vascular resistance, or because it is concentrated and excreted by the kidney, it might have
local effects on renal function that could reduce sodium excretion and cause volume-
dependent hypertension (Ito, 1995). The concentrations of L-NMMA circulating in healthy
subjects are much lower than those of ADMA, but increased concentrations have been
reported recently in patients on dialysis (Mendes-Ribeiro et al., 1996). Of the other putative
uraemic toxins, methylguanidine (MacAllister et al., 1994) and guanidine (Sorrentino et
al., 1997) have been shown to be NOS inhibitors, and it is possible that uraemic plasma
contains a cocktail of NOS inhibitors which act in concert to produce chronic low level
inhibition of NO synthesis (Arese et al., 1995).
There is also evidence for increased production of NO in humans with uraemia. Increased
platelet derived NO has been implicated in the bleeding tendency of uraemia (Noris et al.,
1993). Induction of iNOS in immune cells has been detected in glomerulonephritis, (Kashem
et al., 1996), urinary tract infection, and (Wheeler et al., 1997; Smith et al., 1996) and renal
transplant rejection (Smith et al., 1996). Moreover, nitroryrosine (a putative marker for
elevated NO concentrations) has been observed in tissue from transplanted kidneys following
chronic rejection (MacMillan-Crow et al., 1996).
Endothelin
Circulating plasma concentrations of endothelin are increased in patients with post-ischaemic
renal failure (Tomita et al., 1989; Moore et al., 1992, septic shock (Weitzberg et al., 1991;
Sanai et al., 1995), and renal failure associated with radio contrast media (Marguiles et al.,
1991; Clarke et al., 1997). Plasma concentrations of immunoreactive endothelin are also
increased in patients with chronic renal failure, and are inversely proportional to renal
function (Koyama et al., 1989; Warrens et al., 1990). Selective assays reveal that this increase
is due to marked elevations in plasma endothelin-1 concentrations with little or no change
in big endothelin-1 concentrations (Ferro et al., 1998). Such elevations in mature endothelin-
1, without changes in big endothelin-1, are most likely due to decreased renal clearance of
endothelin-1 (Kohno et al., 1989). Even so, in some cases, circulating endothelin-1
concentrations may reach levels that would allow endocrine actions for the peptide
(Cockcroft & Webb 1989). For example, endothelin-1 concentrations in haemodialysis
patients are positively correlated with arterial pressure (Miyauchi et al., 1991; Tsunoda et
al., 1991), in contrast to essential hypertension (Davenport et al., 1990). In addition to
potential endocrine effects of high circulating endothelin-1 concentrations, local tissue
endothelin-1 generation may also be increased in the kidney of patients with chronic renal
failure, and this might explain the five-fold increase in immunoreactive endothelin in the
urine of patients with chronic renal failure (Ohta et al., 1991). It is possible that increased
urinary endothelin excretion merely reflects a renal tubular homeostatic response to decreased
sodium reabsorption. Plasma immunoreactive endothelin-1 concentrations are increased
after intravenous but not subcutaneous erythropoietin administration (Carlini et al., 1993;
Hand et al., 1995; Portoles et al., 1997).
It is not possible to ascribe definite pathogenic roles to endothelin on the basis of
biochemistry alone, and functional studies of patients with renal disease suggest that activity
of the endothelin pathway is reduced or normal, but not increased. Although the sensitivity of
veins to endothelin-1 is normal in normotensive patients with chronic renal failure, responses
are reduced in hypertensive patients (Hand et al., 1994). This might reflect endothelin receptor
down-regulation secondary to increased concentration of endothelin-1. Patients with chronic
renal failure also exhibit impaired forearm vasodilatation to brachial artery infusion of the
ETA antagonist BQ-123, suggesting reduced endothelin-1-dependent constriction (Hand et
al., 1995a). This could be due to ETA receptor down-regulation, or endothelial dysfunction
leading to decreased tonic ETB receptor-mediated nitric oxide formation. Although regional
vasodilatation following endothelin receptor blockade is attenuated in chronic renal failure,
systemic administration of the ETA/ B receptor antagonist TAK-044 to patients with chronic
renal failure reduces blood pressure by 11% and renal vascular resistance by 10% (Ferro et
al., 1998). However, these patients do not appear to be more sensitive to endothelin-receptor
antagonists than healthy controls (Schmetterer et al., 1998).
THERAPEUTIC POSSIBILITIES
Prostaglandins
Both PGA and prostacyclin have been given to patients with acute renal failure, and although
they caused dilatation, there was no improvement in renal function (Vincenti et al., 1978;
Ladefoged et al., 1970). When used in inflammatory renal diseases, NSAIDs reduce
glomerular filtration and worsen renal function, effects which have discouraged their use in
these diseases (Patrono et al., 1986). It has been suggested that reduced filtration might
slow the progression of renal disease, but this has yet to be investigated. It is unclear if
selective COX-2 inhibitors will offer any advantages over currently available NSAIDs. If
manipulation of the renal prostaglandin pathways has met with limited enthusiasm as a
therapeutic step, it is likely that there are beneficial effects of low-dose aspirin on the systemic
vasculature of patients with renal failure.
NO
Given the evidence for the pathophysiological roles for reduced or increased NO production
in experimental renal disease, there might be therapeutic advantages in increasing or
decreasing the bioavailability of NO in renal disease. Dietary intervention with L-arginine
has been shown to improve renal function in a variety of animal models of acute and chronic
renal disease (Reyes et al., 1994). This is probably a NO-mediated phenomenon, because
the dosing schedules used in these studies avoid the high concentrations of L-arginine that
have non-stereospecific effects. There are no studies of the effects of L-arginine in acute
renal failure in humans. Nor are there any studies of chronic supplementation of L-arginine
in chronic renal disease, although one study of low dose L-arginine given acutely has been
shown to reduce proteinuria in patients with glomerulonephritis excretion (Wolf et al., 1995).
In the systemic vasculature, NO-mediated dilatation might be augmented by provision of
L-arginine to antagonise the possible effects of L-arginine analogues. In patients with chronic
renal failure, low-dose L-arginine (but not D-arginine) improves endothelium-dependent
dilatation of human veins. Whether chronic therapy with L-arginine or NO donors might
augment NO-mediated effects to improve blood pressure control and retard atherogenesis
remains to be determined. In summary, given the available animal and human data there is
a clear rationale for dose-ranging studies of acute and chronic L-arginine supplementation
in human renal disease, to examine effects on renal and systemic vascular function. An
alternative to L-arginine might be to use NO donors, but this approach has yet to be
systematically investigated in animal and clinical renal disease. Selective inhibition of iNOS
has promise as an anti-inflammatory therapy but advances in this area await the development
of useful selective iNOS inhibitors that can be tested in renal inflammation.
Endothelin
The vasodilator and hypotensive effects of endothelin receptor antagonists in both healthy
subjects (Haynes et al., 1994; Haynes et al., 1996) and patients with renal impairment
(Hand et al., 1995a; Ferro et al., 1998) indicate that these agents may be therapeutically
useful in chronic renal failure. In one small study of patients with acute renal failure, BQ-
123 caused renal vasodilatation at doses that did not alter systemic blood pressure, but did
not improve renal function (Soper et al., 1998).
SUMMARY
Endothelial mediators have important physiological roles in the human kidney. NO and
prostaglandins have broadly similar dilator and natriuretic roles, that contrast with the
largely vasoconstrictor and anti-natriuretic effects of endothelins. In certain renal diseases
of humans there is evidence for increased prostaglandin production that contributes to
the maintenance of renal blood flow. In addition, there is evidence for reduced activity of
the NO pathway in the systemic vasculature of patients with chronic renal failure, possibly
due to reduced clearance of NOS inhibitors as renal function declines. However, it is
unclear if the renal NO pathway is abnormal in renal diseases or if the pro-inflammatory
effects of NO might be involved in renal disease. The role of endothelin-1 in renal disease
is complex. In the kidney, there is increased endothelin-1 generation and receptor
expression that could contribute to the progression of renal injury. Systemically, there
are increased circulating concentrations of endothelin-1 caused by impaired renal clearance
of the peptide that might cause hypertension. However, functional studies to date do not
provide evidence for increased endothelin-mediated constriction in renal disease.
Nonetheless it is possible that manipulation of endothelial mediators, either singly in
concert, might form the basis for new therapies in the future. Such therapies will need to
improve renal function or to treat the complications of atherosclerosis, the commonest
cause of death in patients with renal disease.
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Inflammation of the blood vessel wall is responsible for much of the dysfunctional systemic responses of the
circulation during disease states such as septic shock. Most studies exploring the effects of inflammatory signals
on vascular reactivity have been undertaken in animals. However, it is recognised that there is considerable species
variation in the mechanisms of inflammation and that the results of studies in vitro differ from those undertaken
in vivo. This chapter draws together the research that relates to recent work in the field of sepsis and attempts to
unravel some the pathophysiological changes that are occurring within the vascular wall during this process.
Reference is made to much of the human in vitro and in vivo data that relates to this condition and emphasis has
been placed on trying to understand some of the changes occurring in the endothelial and smooth muscle function
during this process of inflammation.
Key words: Inflammation, cytokines, endotoxin, nitric oxide, prostanoids, endothelin

12 Infection and Vascular Inflammation
Kiran Bhagat1 and Timothy W.Evans2

1
Centre for Clinical Pharmacology, University College London, 5 University Street,
London, WC1E 6JJ, UK
2
Department of Critical Care Medicine, National Heart and Lung Institute, Dovehouse
Street, London, SW3 6LY, UK
Until recently, the endothelium was perceived to be a passive, metabolically inert permeability
barrier whose function was primarily to contain blood and plasma. However, endothelial cells
are now recognised as metabolically and physiologically dynamic, playing a primary and
fundamental role in the vascular response to sepsis and systemic inflammation (Figure 12.1).
In addition to responding rapidly (in seconds to minutes) to inflammatory agonists such as
bradykinin and histamine; upon exposure to endotoxin or cytokines, endothelial cells undergo
profound alterations of function that involve changes in gene expression and protein synthesis
(Mantovani et al., 1992; Pober and Cotran, 1990; Introna et al., 1994). The endothelial cell
responds to endotoxin via a direct, lipopolysaccharide binding protein (LBP) and a soluble
CD-14 (sCD-14)-dependent pathway (Kielian and Blecha, 1995); inhibition of this binding
by monoclonal antibodies against CD-14 has been shown to reduce the production of cytokines
by endothelial cells in vitro as well as the hypotension and end organ dysfunction in primate
models of endotoxaemia (Leturcq et al., 1996). Most of the metabolic effects of endotoxin
are mediated by endothelial and smooth muscle production of cytokines. The pro-inflammatory
cytokines such as IL-1, TNF IL-6 and interferon are synthesised by cultured human endothelial
cells in vitro (Pober and Cotran, 1990; Introna et al., 1994).
The primary vasoactive substances released by endothelial cells identified to date are
nitric oxide (NO), the vasoconstrictor peptide endothelins (ETs), and a variety of other
vasoactive substances including the prostanoids released via the cyclooxygenase (COX)
pathway of arachidonic acid metabolism.
The inflammatory response represents the reaction of the vasculature and its supporting
elements to injury, and results in the formation of a protein-rich exudate, provided the injury
has not been so severe as to cause actual tissue destruction. Many of the substances involved
in modulating the acute inflammatory response exert their effects via cytokines. Moreover,
a number of the clinical manifestations of acute bacteraemia or septicaemia are thought to
be mediated by the outer wall of these organisms—exotoxin in the case of Gram-positive
bacteria and endotoxin in the case of Gram-negative bacteria.
ENDOTOXIN AND VASODILATATION
Endotoxin is one of the integral components of the outer bacterial lipopolysaccharide cell
wall of all Gram-negative bacteria (Rietschel et al., 1994), the lipid A component being
responsible for most of the molecule’s toxicity. Lipopolysaccaride is highly conserved and
227
228 Kiran Bhagat and Timothy W.Evans
Figure 12.1 Schematic representation of endothelial/smooth muscle interaction under (top) physiological conditions
and (bottom) during systemic inflammation.
Infection and Vascular Inflammation 229
appears to be essentially invariable and present in all forms of endotoxin (Luderitz et al.,
1966). Several lines of evidence indicate that endotoxin, and in particular lipid A, is the
primary exogenous mediator in the development of vasodilatation associated with Gram-
negative bacterial infections. Experimental studies have shown that injection of endotoxin
results in a constellation of symptoms almost identical to those observed in Gram-negative
vascular inflammation (Braude, 1980). Severe vascular wall inflammation and vasodilatation
occur about 30 min. after injection of endotoxin in dogs and results in eventual vascular
collapse (Snell and Parrillo, 1991).
Leukocytes (as well as other cells) respond to endotoxin by secreting a variety of
cytokines that heighten the defensive responses of the host. Tumor necrosis factor (TNF),
interleukin (IL)-1, and IL-6 are secreted acutely following stimulation of mononuclear
phagocytes with endotoxin (Pugin et al., 1993). These substances induce the acute phase
vasodilatory inflammatory response and prime the immune system for rapid activity. The
profile of cytokines secreted in response to endotoxin is now relatively well documented,
but the molecules involved in the initial recognition of endotoxin are still being discovered.
Recent studies have led to the recognition of at least 3 classes of molecules on leukocytes
(and in some cases endothelial cells) that are receptors for endotoxin. The CD18 molecules
(leukocyte integrins) bind endotoxin and participate in the phagocytic engulfment of
bacteria. The scavenger (acetyl-low-density lipoprotein) receptor recognises free
circulating endotoxin and mediates its uptake and degradation. A third receptor, CD14,
recognises complexes of endotoxin with the serum protein lipopolysaccharide-binding
protein (LBP) (Pearson, 1996; Schletter et al., 1995). CD14 appears to participate in
both the ingestion of, and synthetic responses to, endotoxin because blockade of CD14
with monoclonal antibodies strongly inhibits uptake of endotoxin and secretion of TNF
by human mononuclear cells (Pugin et al., 1993; Wright, 1991). Other endotoxin receptor
molecules have also been proposed but further studies are needed to confirm their
involvement (Wright, 1991).
The CD14 molecule is also present in the soluble form as it is shed from cells (Bazil
and Strominger, 1991). Soluble CD14 (sCD14) can bind and increase endotoxin-induced
activities, such as oxidative burst responses (Schutt et al., 1991) and TNF production
by whole blood cells (Haziot et al., 1994). CD14 may also increase the response to
endotoxin by cells that do not constitutively express this receptor: recent reports have
demonstrated that activation of endothelial cells (which do not have cell-bound CD14
receptors) by endotoxin is mediated by sCD14 which can act as a receptor for endotoxin
for the endothelial cell (Noel, Jr. et al., 1995). The vascular smooth muscle (VSM),
lacks CD14 receptors, but several studies have shown that endotoxin can act directly
on VSM to induce a loss of vascular tone and consequent vasodilatation (Beasley et
al., 1990). Again this seems to be due to activation by sCD-14-endotoxin complex
(Loppnow et al., 1995).
CYTOKINES AND VASODILATATION
Cytokines are small proteins (MW 8–30000 daltons), that possess multiple biological
activities. They are active in low (pico-femptomolar) concentrations and are produced
primarily in response to external stimuli, for example, to endotoxin and exotoxin. Most
pro-inflammatory cytokines are produced primarily in the presence of infection or disease
and contribute to the immune response, inflammation and endothelial cell activation
(Mantovani et al., 1992). The results of many animal and human studies in which endotoxin
has been administered systemically, have characterised the changes in cytokine levels
that occur following injection (Cannon et al., 1990; Hesse et al., 1988; Klosterhalfen et
al., 1992; Michie et al., 1988; Kuhns et al., 1995). During the acute response to endotoxin
administration there is a dramatic rise in 3 pro-inflammatory cytokines—TNFα, IL-1ß
and IL-6 (Hesse et al., 1988; Klosterhalfen et al., 1992; Michie et al., 1988). Sixty minutes
after endotoxin challenge, levels of TNFα have reached their peak, IL-6 levels increase
by 90 min. and IL-1ß (3 by 120 min. TNFα has been shown to be a potent inducer of IL-
1 and IL-6 release (Fong et al., 1989). Moreover, IL-1ß is also able to upregulate IL-6
(Shalaby et al., 1989). These studies were carried out using a bolus injection of endotoxin
and it is possible, however, that the alterations of cytokine release induced by a persistent
infective focus may differ from the changes observed following a bolus injection of
endotoxin.
Clinical studies (Hesse et al., 1988; Waage et al., 1989b; Waage et al., 1989a; Waage et
al., 1987) have demonstrated that TNFα and IL-1ß blood levels are significantly elevated
in patients with endotoxaemia, and that an increase in IL-6 occurs during infectious episodes
in humans (Waage et al., 1989a). However, elevated levels of a particular cytokine in the
systemic circulation may represent the levels required to induce changes in more distal
tissues. Similarly, the lack of detection of a specific cytokine may result either from a lack
of synthesis, the rapid clearance of that cytokine by binding to available receptors in target
tissues, rapid renal clearance of cytokine-soluble receptor complexes, or a lack of assay
sensitivity in detecting physiological levels of specific cytokines. This may explain why
some studies fail to detect significant levels of IL-1ß (or other pro-inflammatory cytokines)
in the systemic circulation following injection of endotoxin (Kuhns et al., 1995).
When injected into experimental animals, TNFα and IL-1ß both induce vasodilation
and shock; and act synergistically when administered together (Dinarello et al., 1989). A
single intravenous injection of TNFα or IL-1ß administered to patients with cancer induces
a sudden fall in blood pressure often requiring treatment (Chapman et al., 1987; Walsh et
al., 1992). Healthy volunteers receiving TNFα respond in a similar fashion (van der Poll et
al., 1990; van der Poll et al., 1992), whilst an infusion of IL-6 into patients results in fever,
chills, and minor fatigue, a significant increase in C-reactive protein, fibrinogen and platelet
counts but no hypotension (van Gameren et al., 1994; Weber et al., 1994; Weber et al.,
1993). The effects of three key pro-inflammatory cytokines (TNFα, IL-1ß, and IL-6) will
be discussed in more detail.
TNFα and Vasodilatation

TNFα is principally a macrophage/lymphocyte-derived cytokine with a broad spectrum of
immunoregulatory, metabolic and pro-inflammatory activities (Beutler et al., 1985; Bazzoni
and Beutler, 1996). TNFα is a trimer with a total molecular weight of 52 kDa that interacts
with 2 receptors—R1 and R2, which mediate cytotoxic and proliferative responses
respectively. These receptors are present on nearly all cell types with the exception of
erythrocytes and unstimulated T-lymphocytes. Although the presence of the receptor appears
to be a prerequisite for a biological effect, there does not seem to be a correlation between
the number of receptors and the magnitude of the response (Bazzoni and Beutler, 1996;
Vassalli, 1992). Soluble TNFα binding proteins have also recently been characterised (Spinas
et al., 1992) There are 2 types, antigenically distinguishable and corresponding to the shedded
extracellular domains of the 2 species of cell-bound receptor. The presence of soluble TNF-
R in the serum may compete and inhibit the binding of TNFα action on cells in addition to
affecting the pharmacokinetics and stability of TNFα (Suffredini et al., 1995). On many
cell types, even in the absence of protein production, TNFα causes the release of arachidonic
acid and consequent secretion of prostanoids (Fiers, 1991). Moreover, treatment of
endothelial cells with TNFα induces excessive production of PGI2 and PGE2, platelet
activating factor (PAF) and nitric oxide (NO) (McKenna, 1990; Lamas et al., 1991), all
potential vasodilators both in vitro and in vivo. The addition of TNFα to many cell types
induces protein synthesis following gene activation. This has been studied in more detail in
endothelial and polymorphonuclear cells, monocytes and lymphocytes; which are the main
targets for circulating TNFα (Fiers, 1991; Old, 1985).
Administration of TNFα depresses endothelium-dependent relaxation in vivo (Bhagat
and Vallance, 1997a), and in vitro TNFα reduces the half-life of mRNA coding for nitric
oxide synthase (Yoshizumi et al., 1993). In addition, in patients with heart failure,
significantly elevated levels of TNF have been documented (Levine et al., 1990) and, in
experimental heart failure, reduced gene expression of endothelial NO synthase (eNOS)
and cyclo-oxygenase-1 (COX-I) activity has been reported (Smith et al., 1996). It is not
known whether COX-II activity contributes to these effects of TNFα, however generation
of free radicals as a by-product of COX activity (Darley Usmar and Halliwell, 1996) might
affect endothelial function, and in studies in animals, the endothelial dilator dysfunction
that occurs during endotoxaemia is significantly restored in the presence of free radical
scavengers (Siegfried et al., 1992).
Phase I studies in cancer patients demonstrated that an acute bolus injection of TNFα
resulted in flu like symptoms within 60–90 min (Saks and Rosenblum, 1992). Chronic
infusion of this cytokine resulted in anorexia and a leucopaenia (57). In healthy volunteers
acute haemodynamic changes were not detected, although a single injection of TNFα elicited
rapid and sustained activation of the common pathway of coagulation, probably induced
through the extrinsic route (van der Poll et al., 1990; van der Poll et al., 1991). Further,
familial differences in endotoxin-induced TNFα release from circulating blood mononuclear
cells following systemic injection of endotoxin have been shown in healthy volunteers (Derkx
et al., 1995). This suggests the possibility of TNFα gene polymorphism which may influence
the inter-individual response to endotoxin challenge.
IL-1ß and Vasodilatation

The IL-1ß gene family is comprised of IL-1a, IL-1ß and IL-1 receptor antagonist (IL-1Ra,
62). Each is synthesised as a precursor protein; the precursors for IL-1ß (pro-IL-1a and proIL-
1ß) have molecular weights of 31 kDa. The proIL-1ß and mature 17 kDa IL-1 ß are both
biologically active whereas the proIL-1ß requires cleavage to a 17 kDa peptide for optimal
biological activity. The IL-1Ra precursor is cleaved to its mature form and secreted like most
proteins. IL-1ß remains cytosolic in nearly all cells, but unlike IL-1ß, IL-1ß is rarely found in
the circulation of inflammatory fluids. There is evidence that IL-1a functions as an autocrine,
intracellular messenger, particularly in cultured endothelial cells and fibroblasts (Elliott et al.,
1994). IL-1ß also remains cytosolic in nonphagocytic cells. In mononuclear cells, however,
between 40–60% is transported out of the cell. Unlike IL-1a, the IL-1ß precursor requires
cleavage for optimal secretion and activity. A fundamental property of IL-1ß, like TNFα, is
its ability to induce gene transcription of its own gene (Dinarello et al., 1987) in addition to a
wide variety of other genes. Cultured endothelial cells exposed to IL-1ß increase the expression
of adhesion molecules, which leads to the adherence of leukocytes to endothelial surfaces.
These treated endothelial cells also increase production of prostaglandins, PAF, NO and
synthesis of other cytokines, all of which may contribute to the acute vasodilatation seen
during the acute inflammatory response. Similarly, IL-1ß inhibits smooth muscle contraction
and this effect appears to be largely dependent on NO production leading to increased guanylate
cyclase activity (O’Neill, 1995; Bankers Fulbright et al., 1996; Beasley and McGuiggin, 1994;
Beasley and Eldridge, 1994). There appear to be at least two defined IL-1ß cell-mediated
receptors. Type I and II. In general, IL-1ß binds to type I and IL-1 to type II (O’Neill, 1995;
Bankers Fulbright et al., 1996). Following receptor binding IL-1ß has been shown to increase
protein phosphorylation in cells, and much effort has been made to identify the protein kinases
responsible. IL-1ß causes rapid induction of a wide variety of genes that encode
proinflammatory proteins as well as cytokines that initiate or augment inflammatory cell
activation. The induction of these genes is regulated by IL-1ß inducible transcription factors
that are members of the immediate-early gene response family, including AP-1 and NF?B.
These transcription factors can be activated within minutes of IL-1ß receptor ligation
independent of de novo protein synthesis. IL-1ß can also induce the synthesis of components
of these transcription factors later in the activation program. The mechanisms responsible
for mediating synergistic functions by interaction of different IL-1ß inducible transcription
factors or other transcription factors is incompletely understood (O’Neill, 1995; Bankers
Fulbright et al., 1996).
Diminished vascular contractility of rat aorta is seen in vitro when the tissue is incubated
with IL-1ß (McKenna et al., 1988; Beasley and McGuiggin, 1994; Beasley and Eldridge,
1994; Beasley et al., 1991). In some studies chronic incubation with IL-1ß results in a
biphasic reduction in contractility (McKenna et al., 1989) suggestive of the same changes
that occur in vivo in animals and healthy volunteers injected with endotoxin. In animal
studies injection of high dose IL-1ß (>1µg/kg) results in acute vasodilatation, decreased
systemic vascular resistance, depressed myocardial function, vascular leak and pulmonary
congestion (Dinarello, 1994). IL-1ß deficient mice responded normally to the systemic
administration of endotoxin with no improvement in terms of mortality when compared to
wild mice. However, the local acute phase tissue response to the inflammatory stimulus is
absent when compared to IL-1ß competent mice (Fantuzzi and Dinarello, 1996). This
suggests that the systemic response to endotoxin may involve other cytokines with
overlapping activities but that IL-1ß appears to play a key role in the local inflammatory
response.
Results from recent studies in humans (Bhagat and Vallance, 1999) have shown that IL-
1ß, can induce functionally significant hyporesponsiveness to vasoconstrictors in humans
and that the effect is mediated by increased NO production. In animal models IL-1ß induces
NO generation through transcriptional up-regulation of iNOS. However in the experiments
performed in this study no functionally active iNOS was detectable and the cause for the
increased NO production was best explained by de novo gene transcription causing an
increase in expression of GTP cyclohydrolase 1 and consequent activation of eNOS by
tetrahydrobiopterin. An implication of the findings is that the endothelium is able to generate
sufficient NO to cause profound vasodilatation even in the absence of expression of iNOS.
The results and those of previous similar studies in cultured cells (Rosenkranz Weiss et al.,
1994; Werner Felmayer et al., 1993; Katusic et al., 1998), suggest that drugs that inhibit
GTP cyclohydrolase I might be of therapeutic use to reverse local or systemic inflammatory

venous dilatation
IL-6
IL-6 (MW 23 kDa) is produced by almost all cell types in response to a variety of different
stimuli including endotoxin or cytokines (such as IL-1ß (Content et al., 1985), and TNFα
(Jablons et al., 1989)). The gene for IL-6 contains consensus sites for ubiquitous transcription
factors such as AP-1, NF-?B, a c-fos serum-responsive element, and a cyclic AMP-responsive
element (Scholz, 1996). A number of studies have shown IL-6 to be important in the
regulatory production of acute phase proteins during an inflammatory response (Rusconi
et al., 1991; Helfgott et al., 1989; Ulich et al., 1991; Furukawa et al., 1992). In cell culture,
IL-6 does not appear to affect the prothrombotic or proinflammatory effects of IL-1ß on
vascular cells and it has been suggested that the key role that of IL-6 is in activating T and
B lymphocytes (in addition to the systemic production of acute phase reactants). The
production of IL-6 by endothelial cells supports the notion that these cells are involved in
immunological pathways and in the regulation of the acute-phase response. Incubation of
vascular tissue with IL-6 appears to cause an impairment on contractility in several animal
studies (Ohkawa et al., 1995); however, in vitro studies using human vessels fail to produce
any changes in vessel tone (Beasley and McGuiggin, 1994). This may be due to the absence
of circulating cells or intraluminal factors that may be important for IL-6 action in vivo.
At the time of writing, no trials looking at the effects of IL-6 receptor blockade during
endotoxaemia (either in healthy volunteers or during infective states) have been published,
but in both the clinical trials using anti-TNF or IL-1Ra high circulating concentrations of
IL-6 predicted a worse outcome irrespective of treatment (Derkx et al., 1995; Fisher, Jr. et
al., 1994). Recent investigations have also suggested a relationship between blood
concentrations of IL-6 and poor outcome during several inflammatory conditions, including
acute myocardial infarction (Neumann et al., 1995), major surgical procedures (Scholz,
1996) and Kawasaki disease (Furukawa et al., 1992). It remains to be determined whether
IL-6 antagonism in other non-infective inflammatory diseases such as unstable angina and
myocardial infarction confers any benefit in terms of outcome.
NO Release in Infection
There is considerable interest in the role of NO as a mediator of physiological and
pathophysiological vasodilation. NO has been shown to be synthesized in vitro from the
semi essential amino acid L-arginine by the membrane bound NO synthases (NOS), a process
that can be inhibited by L-arginine analogues such as NG-monomethyl-L-arginine (L-
NMMA). Several distinct NOS isoforms have been identified. The enzyme is expressed
constitutively in endothelium and neural tissue (e, nNOS) and is inducible in a variety of
cell types (iNOS). e/n and iNOS are calcium and calmodulin dependent and independent
respectively.
In healthy vessels, production of NO in the cardiovascular system occurs mainly from
endothelial cells expressing eNOS. However, endotoxin, cytokines including IL-1ß and
TNFα, and products of Gram-positive bacteria induce the expression in endothelial and
smooth muscle cells of iNOS. Induction of iNOS involves protein synthesis and is inhibited
by glucocorticoids (Rees et al., 1990). Interestingly the production of NO synthase by the
constitutive NOS appears to be inhibited following induction of the inducible NOS and this
effect appears to be mediated by a decrease in the stability of the mRNA encoding for
constitutive NOS message (Yoshizumi et al., 1993). Thus whereas in the healthy vessel the
endothelium is the major vascular source of NO, during inflammation the whole vessel
synthesises this mediator.
Patients and animals with sepsis lose peripheral vascular tone and the responsiveness of
vessels to constricting agents both in vitro and in vivo is diminished (Lorente et al., 1993).
The incubation of bovine aortic endothelial cells with lipopolysaccharide causes a rapid
release of an NO-like factor (Salvemini et al., 1990; Kilbourn and Belloni, 1990), and in
patients with sepsis the level of NO metabolised in plasma is significantly elevated. Infusion
of NOS inhibitors in such cases can lead to a rapid and reproducible rise in systemic vascular
resistance where other pressor substances are ineffective (Ochoa et al., 1991; Petros et al.,
1991). It seems likely that the synthesis and release of NO is stimulated by this inflammatory
process (Figure 12.2). Endotoxin leads to the induction of an iNOS in endothelium and
underlying vascular smooth muscle as well as myocardium (Rees et al., 1990; Radomski et
al., 1990; Fleming et al., 1991a). TNFα and IL-1ß can also stimulate the expression of
iNOS in both endothelium and vascular smooth muscle. Patients treated with IL-2
chemotherapy excrete high levels of NO metabolites suggesting that induction of NO
synthesis occurs in response to this treatment. Interestingly, TNFß can also inhibit NO
release stimulated in isolated pulmonary vessels by specific agonists (eg acetylcholine and
bradykinin) although basal NO release is unaffected.
The possibility of a two stage release of NO from the blood vessel wall during sepsis
has been suggested. In isolated, endotoxin-treated rat vein and pulmonary arteries, NOS
inhibitors reverse the vascular hyporesponsiveness to phenylephrine. Moreover, NO-
mediated hyporeactivity to noradrenaline starts within 60 minutes in a rat model of sepsis
in vivo and is therefore too rapid to be explained by the induction of iNOS. Information
regarding the latter is presently uncertain, although experiments in animal models suggest
that mRNA encoding for iNOS production may be detectable as early as 20 minutes after
the intraperitoneal injection of endotoxin (Liu et al., 1997). The endothelium also seems
to respond immediately to the septic insult by releasing NO produced by the constitutive
enzyme, eNOS. However, other studies have suggested down regulation of mRNA
expression encoding for eNOS in parallel with up-regulation of iNOS mRNA (Liu et al.,
1997). The hypothesis that endotoxin leads to an increase in NO release from endothelium
causing an early loss of vascular responsiveness in vivo therefore remains unproven.
However, from about 3h following an endotoxic insult there is massive NO production
attributable to iNOS, most probably induced in vascular smooth muscle (Fleming et al.,
1993). There is also evidence that endothelium is required for the NO response to be
maximal. Thus, NO removal has been shown to cause significant delay in the onset of
vascular hyporesponsiveness (6 hrs compared with 4 hrs) and reduced the sensitivity of
rat aorta exposed to lipopolysaccharide in vitro (Fleming et al., 1991b; Fleming et al.,
1993). Selective inhibitors for iNOS are now available and are the subject of intense
investigation (see below).
ETs in Infection
In 1988 an endothelially-derived vasoconstrictor was cloned and sequenced following its
isolation from the culture medium of porcine aortic endothelial cells and termed endothelin
Figure 12.2 Activation of humoral and cell-mediated pathways by endotoxin resulting in inflammation and tissue
damage. Abbreviations: endo-derived, endothelium derived; PMN, polymorphonuclear leucocytes; Tx,
thromboxane; PGs, prostaglandins; LTs, leukotrienes.
(ET) (Yanagisawa et al., 1988). The substance was found to elicit a slow onset, sustained
contraction of isolated arteries from many different species. Three similar but distinct ET-
related genomic loci have now been identified encoding for three similar but distinct ET
molecules (ET-1, ET-2) (Inoue et al., 1989). Three ET receptor subtypes probably exist
although so far only two have been cloned and expressed (Arai et al., 1990). ETA has the
highest affinity for ET-1 compared with the other ET’s and although it has wide spread
expression in human in particular vascular smooth muscle, this does not include endothelial
cells. ETB is non-selective, binding all three ET’s which are of equipotent in displacing
radiolabelled ET-1. ETB expression is also wide spread including endothelial cells. ET’s
probably work by increasing intra-cellular free calcium, activating phospholipase C leading
to increases in inositol trisphosphate and diacylglycerol synthesis. Both are implicated in
the initial rise in intra-cellular free calcium concentrations and probably underlie the initiation
of ET-1 induced vascular contraction. Protein kinase C is also implicated in a second-
messenger system mediating ET-1-induced contraction. ETs are not stored but are
synthesised de novo in the endothelium. Endothelial cells exposed to agents such as
adrenaline and thrombin express ET-1 mRNA. ET-1 production has also been demonstrated
both a the mRNA transcription level and at that of protein release in response to angiotensin
2, thrombin and transforming growth factor beta. Mechanical shear stresses, and hypoxia
also induce ET-1 production, suggesting that it has a role in modulating local blood flow in
response to changes in these indices. Lastly, platelets stimulate expression of ET, mRNA
and ET biosynthesis in cultured endothelial cells (Yoshizumi et al., 1989; Kourembanas et
al., 1991; Elton et al., 1992; Ohlstein et al., 1991).
ET-1 is a potent vasoconstrictor in humans and many other species. ET-1 and ET-3-
induced contraction of rat pulmonary arteries is enhanced by endothelial removal and NO
release has been demonstrated in rat mesentery and response to ET, an effect inhibited by
NO blockade. ET-3 is a more potent vasodilator that ET-1 probably because the receptor
involved on the endothelium is of the ETB type which has equal affinity for all ET’s. By
contrast, the ETA (vasoconstrictor) receptor predominates in vascular smooth muscle and
has a higher affinity for ET-1 (Hirata et al., 1993). Most species demonstrate a characteristic
bi-phasic response to ET infusion, with initial transient hypotension followed by sustained
hypertension. The former may be partially mediated by NO (Karaki et al., 1993). Specifically,
ET-3 is a powerful vasodilator in isolated rat lungs under prior conditions of hypoxic
vasoconstriction, although ET-2 and ET-2 are both powerful pulmonary and coronary
vasoconstrictors (Karaki et al., 1993). ET-1 has inotropic and chronotropic effects on cardiac
muscle and can also induce proliferation in cultured vascular smooth muscle, suggesting a
role in remodelling.
Under inflammatory conditions, ET’s may be important mediators of vascular tonic
responses. Specifically, ET release in response to endotoxin has been confirmed in in vivo
and in vitro, and in endothelial cell cultures in response to a variety of cytokines and free
radical species (Sugiura et al., 1989). ET levels increase during endotoxaemia in many
animal models and are elevated possibly in parallel with indicators of disease severity in
patients with sepsis (Weitzberg et al., 1991; Voerman et al., 1992; Pittet et al., 1991).
Although the release and high circulating levels of such a potent vasoconstrictor substance
would be expected to antagonise the characteristic refractory hypotension (thought to be
mediator by NO) that is seen in sepsis and related syndromes, this does not seem to occur.
It is possible that a complex interaction between ET’s and NO explains this paradox.
COX Products in Infection

The endothelium also produces various prostanoids via the COX pathway of arachidonic
acid metabolism, principle among which is the vasodilator prostacyclin which induces
smooth muscle relaxation via the activation of adenylate cyclase thus increasing intracellular
cyclic AMP levels. Cyclooxygenase inhibition augments certain vascular reflexes including
hypoxic pulmonary vasoconstriction, implying that vasodilator prostoglandins may modulate
vascular tone during hypoxaemia (Hales et al., 1978; Weir et al., 1976). Moreover, cytokines
can stimulate prostanoid release, the vasodilator action of which is augmented by the presence
of endothelium at least in the porcine coronary circulation. Thromboxane A2 and related
endoperoxides are potent vasoconstrictors which are also capable of inducing capillary
permeability. ET-mediated release of certain prostanoids including prostacyclin and
thromboxane has been demonstrated. Indomethacin pre-treatment of isolated vascular
preparations in certain species potentiate ET-induced contraction and even increases ET-3
induced ET-1 formation in cultured endothelial cells, implying that prostacyclin is able to
inhibit ET production.
Arachidonic acid is formed from cell-membrane phospholipid when the membrane is
disturbed; this disturbance leads to the activation of phospholipase A2 (Flower and Blackwell,
1976). Arachidonic acid is then available for the formation of secondary metabolites.
One class of products of the arachidonic acid cascade are the prostaglandins (PGs).
The rate limiting enzyme for this prostanoid pathway is COX (Hemler and Lands, 1976).
At least 2 isoforms of COX are now known to exist, one which is present as a normal
constituent of healthy endothelium (COX-I) and the other which is induced in response
to endotoxin and cytokines (COX-II (Hla and Neilson, 1992). The constitutive form of
COX is thought to mediate many physiological functions. Its activation leads, for instance,
to the production of prostacyclin which when released by the endothelium is anti-
thrombogenic and vasodilatory (Moncada et al., 1976). Both the smooth muscle and the
endothelium have been shown to express COX-I and to generate both constrictor and
dilator prostanoids. While most cells have the capacity to generate many different
prostanoids there is some selectivity, with platelets and macrophages producing
predominantly thromboxane A2 (TXA2) and endothelial cells PGI2. PGD2 is the principal
prostanoid produced by mast cells and PGE2 by the microvasculature (Moncada and Vane,
1978b; Moncada and Vane, 1978a).
COX-II is induced in many cells including the endothelium and smooth muscle by pro-
inflammatory stimuli (O’Neill and Ford Hutchinson, 1993). Its induction is inhibited by
glucocorticoids and associated with de novo proteins synthesis. Ferreira and others (1993)
showed that using specific antisera and COX inhibitors that IL-1 is a key cytokine for the
release of COX-II metabolites. The role of PGs at the site of inflammation are multiple, but
their major specific action is one of vasodilatation. This is attributed particularly to PGE2
and PGI2. Alone these agents produce little or no vessel leakage but in combination with
substances which increase vessel permeability, they markedly potentiate the formation of
the resultant oedema (Williams and Morley, 1973). A role for this enzyme and its products
in the pathogenesis of sepsis is supported by the finding that COX inhibitors restore blood
pressure in certain animal models (Parratt and Sturgess, 1974).
In vivo human studies in which endotoxin has been injected as a bolus dose have
noted acute activation of the kallikrein-kinin system and changes in the circulating
prostanoid levels (DeLa Cadena et al., 1993). Prior administration of a COX-inhibitor
markedly reduced the acute pyrexia and malaise, but did not affect the early or late
cardiovascular and haemodynamic changes associated with endotoxaemia (Martich et
al., 1992; Godin et al., 1996).
STUDIES IN HUMANS
Investigation of the initial vascular response to sepsis in humans is difficult because of practical
problems in studying patients at the onset of sepsis. Animal models based on the administration
of endotoxin (or cytokines) have been used to study these early vascular events but as discussed
above, these studies are limited by variation in species and end organ damage to endotoxin
(Redl et al., 1996). The haemodynamic alterations that occurred in normotensive and
hypertensive subjects following injection of endotoxin were first documented in 1945, and
demonstrated clearly the arterial and venous dilatation that occurs in endotoxaemia in humans.
Similar and more extensive observations were made subsequently, comparing humans with
other animals (Gilbert, 1960), and a more systematic study of the metabolic and haemodynamic
changes was made by Suffredini and others in 1989. These investigators studied the temporal
response of cytokine elaboration that occur after endotoxin administration as well as
documenting the haemodynamic changes that ensued. Since then several studies looking at
the systemic effects of cytokine administration on the cardiovascular system have also been
performed (Bhagat et al., 1996b; Bhagat and Vallance, 1997b; van der Poll et al., 1995; Bhagat
et al., 1996a). Pharmacological studies have looked at the effects of prior administration of
steroids, NSAIDs, cytokine antagonists and other agents on the response of the cardiovascular
system to the effects of endotoxin and cytokines. However, such studies are limited by several
factors associated with performing studies in healthy volunteers. Thus, administration of any
systemic inflammatory agent into normal subjects is always constrained by the amount of
active agent that can be safely administered. Furthermore, systemic administration of
endotoxin or cytokine (or any other agent) evokes a systemic neurohumoral reflex response
that confounds any attempt to study the mechanisms of changes occurring in the vascularure.
To explore the cellular and mechanistic events that occur (in vivo) as a direct result of the
local interaction of the inflammatory agent with the vessel wall, experimental models have
now been developed that remove the systemic component of the acute inflammatory event
(Bhagat and Vallance, 1997b).
MANIPULATION OF THE INFLAMMATORY RESPONSE
A variety of interventions have been introduced experimentally (and to a lesser extent

clinically) of late designed to manipulate the inflammatory response to sepsis. Essentially,
the treatment of established sepsis and associated syndromes with or without multiple organ
system failure has achieved only limited success, treatment centering largely around
circulatory support and attempts to maximise tissue oxygenation. Treatment strategies have
now diversified and trials of non-selective NOS inhibitors are underway. The potential value
of these agents is great, particularly if specific inhibition of iNOS is possible since it may
allow basal NO production, which is probably important in the normal regulation of blood
flow in the micro-circulation to continue unimpeded. Aminoguanidine has proved to be
one such selective agent in animal models (MacAllister et al., 1994; Tilton et al., 1993).
Inhaled nitric oxide is now in wide spread use as a means by which selective pulmonary
vasodilatation may be achieved in patients with severe lung injury. The short in vivo half
life of the gas, coupled to its delivery only to ventilated alveolar units results in a selective
reduction in PVR, reduced shunt fraction and increased oxygenation (Rossaint et al., 1993).
In certain animal models of sepsis, pre-treatment with steroids has been beneficial
although in clinical investigations high dose steroid treatment has shown no benefit in
reducing mortality in patients with established septic shock, neither have they been shown
to be beneficial either as treatment or prophylaxis in patients with severe lung injury
(Anonymous, 1987; Bone et al., 1987). Infusion of vasodilator prostanoids has been tried
both as treatment for ARDS and also to reduce mortality in patients with sepsis to little
effect. Several survival rates have been improved in septic animals treated with COX
inhibition, but the clinical effects of these agents have been disappointing (Holcroft et al.,
1986; Melot et al., 1989; Bone et al., 1989). Antioxidants, particularly n-acetylcysteine can
neutralise oxygen free radicals and improve gas exchange haemodynamics and survival in
animal models (Bernard et al., 1984). Preliminary studies in patients have been encouraging.
Antibodies directed against adhesion molecules are also in pre-clinical trials. Anti-ET
antibodies and ET receptor antagonists would also theoretically be useful in these
circumstances, in that they have been shown mainly in models of ischaemia to reduce infarct
size following coronary artery ligation with reperfusion in rats.
ACKNOWLEDGEMENTS
Work supported by the British Heart Foundation.

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The vascular endothelial cell monolayer is likely to play a major role in the maintenance of healthy pregnancy.
Through enhanced synthesis of vasodilator prostaglandins and nitric oxide the endothelium is now considered to
contribute to vasodilation of many vascular beds, and so to accommodation of the greatly expanded plasma volume
without elevation of blood pressure. Because of ethical considerations, investigation of endothelial function in
human pregnancy necessarily has been less extensive than in non-pregnant subjects, and much emphasis has been
placed on animal studies. This review concentrates upon human pregnancy and attempts to bring together the
confusing and often disparate literature. The interesting possibility that synthesis of nitric oxide in pregnancy
may be controlled by circulating estrogens is discussed, together with novel suggestion of a vasodilatory role for
an endothelium derived hyperpolarizing factor. Preeclampsia undoubtedly originates in the feto-placental unit,
but dysfunction of the maternal vascular endothelium in pre-eclampsia is now so widely reported that the maternal
preeclamptic syndrome is considered to be a disease of the endothelium. The evidence for maternal endothelial
cell activation and reduced endothelium dependent dilation of the maternal arteries is reviewed and various
hypotheses to explain this phenomenon are addressed. More importantly, consideration is given to the possible
therapeutic interventions which may offer endothelial protection and so amelioration, or prevention, of this life-
threatening disorder.
Key words: Pregnancy, thrombosis, preeclampsia, ADMA, oestrogen, EDHF, LDL.

13 The Endothelium in Human Pregnancy
Lucilla Poston1 and David J.Williams2

1
Department of Obstetrics and Gynaecoloy, GKT, St. Thomas ‘Hospital, Lambeth
Palace Road, London, SE1 7EH, UK
2
Department of Obstetrics and Gynaecology, Imperial College School of Medicine,
Chelsea and Westminster Hospital, Fulham Road, London, SW10 9NH, UK
INTRODUCTION
During healthy human pregnancy, the peripheral circulation of the mother undergoes
profound vasodilatation. Cardiac output increases by 50% secondary to the fall in peripheral
vascular resistance (Figure 13.1), Combined with an increase in circulating blood volume
these changes are geared towards increasing blood flow to the developing fetus. Blood
flow to several maternal organs also increases dramatically during pregnancy. For example,
blood flow to the kidneys goes up by 80%, to the skin of the hands and feet by over 200%,
and most importantly, to the uterine arteries by 1000%. Conversely, blood flow to the
maternal liver does not change, whilst that to the brain actually falls. Unravelling the
mechanism of these widespread, but heterogeneous vascular changes in human pregnancy
has proved challenging.
Figure 13.1 The fall in total peripheral vascular resistance (TPVR; squares) is maximal by approximately 16
weeks gestation and coincides with the rise in cardiac output (diamonds). Measured by Doppler and cross-sectional
echocardiography at the aortic, pulmonary and mitral valves. Adapted with permission from Robson, S.C., Hunter,
S., Boys, R.J. et al. (1989) American Journal of Physiology, 256, H1060–H1065.
247
248 L.Poston and D.J.Williams
The study of pregnant women is difficult because of the unknown, but critical vulnerability
of a developing fetus to pharmacological manipulation. Many investigators have therefore
concentrated upon gestationally related changes in cardiovascular function of a variety of
animals—with frequently conflicting results. There is compelling evidence for a gestational
increase in vasodilator (and therefore anticoagulant) activity of the endothelium. Yet coupled
to this, is the fact that on balance human pregnancy is a prothrombotic state. Furthermore,
there is controversy concerning the proposal that the corollary to ‘upregulation’ of the
endothelium in healthy pregnancy is dysfunctional endothelium in pre-eclampsia. Animal
studies are of little use in the study of pre-eclampsia—a disease unique to humans in which
many of the normal vascular responses to pregnancy appear to fail. This chapter attempts to
summarise the diverse and often complicated literature which describes the role of the
endothelium in the cardiovascular changes of healthy human pregnancy, and why pregnant
women seem particularly vulnerable to diseases in which endothelial cell activation is central
to their pathophysiology.
THE CARDIOVASCULAR SYSTEM IN HEALTHY PREGNANCY
Cardiovascular changes have been recognised as early as five to six weeks after the last
menstrual period i.e. three to four weeks after conception (Robson et al., 1989; Chapman
et al., 1998). At this time an increase in heart rate and ventricular function combine with a
fall in total peripheral vascular resistance and increase in plasma volume to significantly
increase maternal cardiac output (Robson et al., 1989; Brown and Gallery, 1994). This
trend persists until 24 weeks gestation by which time cardiac output has increased by
approximately 45%.
Arterial dilatation in the first trimester creates a relatively underfilled state associated
with a fall in blood pressure (MacGillivray, Rose and Rowe, 1969; Redman, 1995). Renal
vasodilatation, which may even precede peripheral vasodilatation (Davison, 1984), leads
to a rise in effective renal plasma flow (ERPF) up to 80% above pre-pregnancy levels by
the end of the second trimester (Dunlop, 1981). The glomerular filtration rate (GFR)
increases by 50% (Davison and Baylis, 1995). Despite the increase in renal blood flow,
the vasodilated, but relatively under-filled peripheral and renal vasculature is probably
partly responsible for upregulation of the renin-angiotensin-aldosterone (RAA) axis
(Schrier and Briner, 1991; Brown and Gallery, 1994), although hormonal influences
undoubtedly play a role. Oestrogens are well known to increase renin substrate—
angiotensinogen (Schunkert, Danser, Hense et al., 1997). Indirect assays of plasma renin
activity (PRA) during pregnancy, in the presence of elevated angiotensinogen, invariably
report greater levels of angiotensin I and therefore higher levels of PRA (Brown and
Gallery, 1994). However, postmenopausal women on oestrogen replacement therapy
(HRT) have suppressed renin levels as estimated using a specific monoclonal renin
antibody, (despite elevated angiotensinogen levels) compared with those not on (HRT)
(Schunkert et al., 1997). It is possible therefore, that in normal pregnancy elevated
circulating oestrogen levels will stimulate angiotensinogen production, but circulating
renin levels will be comparatively suppressed.
As a result of avid renal sodium and water retention, total extracellular fluid volume
gradually increases by 6–8L with a relatively greater increase in intravascular compared
with interstitial fluid volume (Brown and Gallery, 1994). At 32 weeks gestation plasma
The Endothelium in Human Pregnancy 249
volume reaches a maximum of 40% (1.2L) above pre-pregnancy levels (Gallery, Hunyor
and Gyory, 1979). Furthermore, plasma volume expansion—a characteristic of healthy
pregnancy, could enhance NO generation (Calver et al., 1992). Increased plasma renin
activity generates higher circulating levels of angiotensin II (ANG II), associated with
reduced binding of ANG II to platelets (Baker, Broughton-Pipkin and Symonds, 1992) and
a reduced presser response to exogenous ANG II (Gant et al., 1973). However, down-
regulation of ANG II receptors cannot account for the attenuated vasopressor responses
also found with exogenous administration of vasopressin and noradrenaline (Chesley, 1978;
Williams et al., 1997). Increased activity of vasodilator substances could play this role and
are discussed in detail below.
COAGULATION
Endothelium Derived Clotting Factors

In anticipation of haemorrhage at childbirth, normal pregnancy is characterised by low
grade, chronic intravascular coagulation within both the maternal and utero-placental
circulation (Letsky, 1995). There is evidence for increased levels of clotting factors, especially
fibrinogen (Bonnar, 1987) and depression of fibrinolysis (Kruithof, Tran-Thang, Gudinchet
et al., 1987), although more recent studies suggest the procoagulant state of normal pregnancy
is compensated by increased fibrinolysis (Sorensen, Secher and Jespersen, 1995). The
endothelium is directly involved in promoting a procoagulant state in healthy pregnancy.
During the third trimester, plasma levels of endothelium derived von Willebrand factor are
elevated, promoting coagulation and platelet adhesion (Sorensen et al., 1995). Furthermore,
there is a gestational increase in endothelium production of plasminogen activator inhibitor
(PAI-1) and tissue plasminogen activator (t-PA), with the effect of both inhibition and
promotion of fibrinolysis, respectively. The t-PA to PAI-1 ratio remains unchanged in healthy
pregnancy (Sorensen et al., 1995). Thrombin generation is also increased in normal
pregnancy, as are circulating levels of fibrin degradation products (FDP) (de-Boer, et al.,
1989; Sorensen et al., 1995), although the ratio of thrombin to FDP remains unchanged.
The procoagulant state of the endothelium does therefore appear to be compensated by
upregulation of the fibrinolytic system (Bremme et al., 1992; Sorensen et al., 1995).
Nonetheless, the risk of thromboembolism increases six fold during pregnancy (Royal
College of General Practioners, 1967) and is the most common cause of maternal death in
the UK (Department of Health, 1998). Factors other than a procoagulant endothelium
contribute to the increased incidence of thromboembolism. In late pregnancy, the gravid
uterus partialy obstructs the inferior vena cava, causing venous stasis in the lower limbs.
Women who have Caesarean sections are also more vulnerable to thromboembolism.
Furthermore, occult thrombophilias often present clinically for the first time during
pregnancy.
NITRIC OXIDE
There is much evidence from animal studies to suggest that increased activity of the L-
arginine—NO pathway contributes to the generalised vasodilatation and attenuated response
to exogenous vasoconstrictors characteristic of pregnancy (Chu and Beilin, 1993; Conrad

et al., 1993; Weiner, Knowles and Moncada, 1994). Conversely, inhibition of nitric oxide
synthase in pregnant rats abolishes the refractoriness to vasopressors and produces elevation
of the blood pressure and proteinuria (see later) (Yallampalli and Garfield, 1993; Allen et
al., 1994). Assessment of the L-arginine—nitric oxide pathway in human pregnancy and
pre-eclampsia has proved more challenging. Different methodologies and conflicting results
present a confusing picture.
Normal Human Pregnancy
Cyclic guanosine monophosphate (cGMP)

The cyclic nucleotide, cGMP acts as a second messenger for NO and has been used as a
surrogate marker for NOS activity. Both plasma and urinary cGMP levels, increase in
pregnant rats coincidental with the gestational increase in renal blood flow (Conrad and
Vernier, 1989). The same group also showed that renal vasodilatation and hyperfiltration
in the pregnant rat is abolished by inhibition of nitric oxide synthase (Danielson and
Conrad, 1995).
In human pregnancy, there is agreement that urinary concentrations of cGMP increase
early in pregnancy and remain elevated until term (Kopp, Paradiz and Tucci, 1977; Chapman
et al., 1998). Cyclic-GMP clearance increases as early as the sixth week of gestation, in
parallel with a fall in plasma cGMP levels, that remain low throughout pregnancy (Chapman
et al., 1998). Others have found slight or significant increases in plasma cGMP during
normal pregnancy, remaining high until term (Schneider et al., 1996; Boccardo et al., 1996).
Boccardo et al. (1996) found cGMP levels and [3H]L-citrulline production within platelets
were similar between pregnant and non-pregnant women, suggesting platelets do not
contribute to excessive NO production in pregnancy.
Cyclic GMP concentrations in plasma or urine are only an indirect indication of NOS
activity as responses to atrial natriuretic peptide (ANP) are also mediated through cGMP.
In a meta-analysis of 53 studies it was concluded that the circulating concentration of ANP
did not rise until the third trimester (Castro, Hobel and Gornbein, 1994). However, this is
long after the increase in urinary cGMP, which coincides with the gestational increase in
renal blood flow and glomerular filtration rate (Dunlop, 1981; Roberts, Lindheimer and
Davison, 1996). It is possible that increased NOS activity has a role in renal vasodilatation
during healthy human pregnancy, but it cannot be concluded from the measurement of
cGMP alone that increased NOS activity plays a role in the gestational fall in systemic
vascular resistance.
Oxidation Products of Nitric Oxide; Nitrite and Nitrate

Several investigators have measured the serum concentration of nitrite (NO2-) and nitrate
(NO3-), or their product, NOx, during healthy pregnancy. However, most have ignored the
confounding problem that concentrations of these NO metabolites are sensitive to dietary
nitrogen intake. Not surprisingly, studies of normotensive pregnant women on uncontrolled
nitrogen diets have shown either increased (Seligman et al., 1994) or unchanged (Curtis,
Gude, King et al., 1995; Smarason et al., 1997) plasma NO metabolites in comparison with
non-pregnant women. In one study, plasma NOx levels were measured after a 12–15 hour
fast, and found to be significantly elevated in normotensive pregnancy (from before 12

weeks gestation until term) compared with non-pregnant women (Nobunaga, Tokugawa
and Hashimoto et al., 1996). In another carefully controlled study, guanidino [N15]L-arginine
was infused into five healthy pregnant volunteers after being on a nitrate free diet for the
preceeding week and a 12 hour fast pre-infusion. Arginine flux and nitrite/ nitrate pool
turnover were higher in early compared with late pregnancy, suggesting NO production is
higher in early pregnancy (Goodrum, Saade, Jahoor et al., 1996).
Nitric Oxide Synthase Activity in Vivo

In vivo functional studies provide the most compelling evidence that NO synthase is
upregulated in the maternal circulation during normal pregnancy. Infusion of the NO synthase
inhibitor, L-NMMA, into the brachial artery caused a greater reduction in hand blood flow
of pregnant compared with non-pregnant women (Williams et al., 1997) (Figure 13.2). The
increased efficacy of L-NMMA was observed during early pregnancy (9–15 weeks), a time
when there was not yet a measurable increase in hand blood flow. This suggests that a
mechanism, other than shear stress, mediates the gestational increase in NO generation.
Furthermore, in late pregnancy (36–41 weeks), L-NMMA returned the elevated hand blood
flow back to non-gravid levels, implicating a major role for NO in the peripheral
vasodilatation of healthy pregnancy (Figure 13.2).
L-NMMA, has also been shown to induce a non-sustained venoconstriction in hand
veins of healthy women in the early puerperium, but not in the same women 12–16 weeks
post-partum (Ford, Robson and Mahdy, 1996). Normally, in the non-gravid state, infusion
of L-NMMA, at a dose that maximally inhibits bradykinin, does not produce venoconstriction
of hand veins (Vallance, Collier and Moncada, 1989). Isolated endothelial cells from hand
veins of pregnant women have been shown to respond to adenine triphosphate (ATP) with
a large transient increase in intracellular Ca2+ (Mahdy et al., 1998). This response was
significantly greater in endothelial cells isolated from the hand veins of healthy pregnant
women compared to non-pregnant and pre-eclamptic women (Mahdy et al., 1998). However,
the extrapolation of evidence from the venous circulation to peripheral vascular control
must be interpreted with caution.
Nitric Oxide in the Resistance Vasculature of the Maternal Circulation

Much of the evidence favouring an increase in endothelial NO synthesis in the vasculature
in pregnancy is derived from studies of small arteries in animals, in which significant
reduction in the EC50 for ACh or methacholine has repeatedly been observed. Although
indicative of enhanced NO synthesis, conclusive proof is not always available as selective
inhibition of NOS has often not been attempted. The reduced responsiveness to constrictor
agonists observed in some small artery preparations from pregnant animals has, using NOS
inhibitors, sometimes been attributed to enhanced NO synthesis. Perhaps the most convincing
evidence for an increase in NOS activity comes from a study of small rat arteries which has
shown an increase in expression of eNOS in pregnancy (Xu et al., 1996). These confusing
and often conflicting studies of NO in the vasculature in animal pregnancy have recently
been covered in considerable detail by a comprehensive review (Sladek et al., 1997).
Limitation in the availability of human tissue is an obvious drawback to investigation in
human pregnancies. However, small arteries may be obtained from subcutaneous fat,
Figure 13.2 Response to brachial artery infusion of L-NMMA (dashed line) and noradrenaline (NE; solid line) on
hand blood flow for nonpregnant (A), early pregnant, (9–15 weeks gestation) (B), and late pregnant (36–41 weeks
gestation) subjects (C). D: area under dose response curve for noradrenaline was subtracted from area under dose
response curve for L-NMMA, for each individual in each group. Response to L-NMMA increases relative to
noradrenaline as pregnancy progresses. Difference between late pregnant and nonpregnant groups was significant
(*P=0.0089). With permission from Williams, D.J., Vallance, P.J.T., Neild, G.H. et al. (1997) American Journal
of Physiology, 272, H748–H752.
omentum and myometrium by biopsy at caesarean section. The development of a variety of

techniques for reproducible investigation of small artery tension and diameter has greatly
facilitated studies in human tissue, as small biopsies may provide adequate material for
experiment. The methods most frequently used have been small vessel wire myography
and small vessel perfusion myography. The wire myograph (Mulvany and Halpern, 1977)
is a commercially available apparatus which enables the measurement of isometric tension
in arteries as small as 150 µm internal diameter. In the perfusion myograph (Halpern et al.,
1984), the small artery is mounted between two fine glass cannulae and a video dimension
analyser used to ‘track’ the movement of the vessel wall as the artery contracts or relaxes.
This instrument has the advantage that pressure transducers enable the artery to be set to
and maintained at a given pressure and the provision of flow pumps also enables investigation
of flow mediated responses.
The blood flow to the skin is greatly enhanced in human pregnancy and investigation of
this circulation may therefore provide insight into gestationally related mechanisms of
dilatation. Small subcutaneous arteries can be obtained by removal of biopsies of
subcutaneous fat from consenting women at caesarean section. The first study of these
arteries, using the wire myograph, investigated responses to ACh in arteries of mean internal
diameter 250–300 µm and showed that relaxation to ACh was no different between arteries
from pregnant women and those from non-pregnant women obtained during routine
abdominal surgery (McCarthy et al., 1994). Interestingly, the NO synthase inhibitor, L-
NMMA failed to completely inhibit relaxation to ACh, and indomethacin had little effect.
The residual relaxation to ACh in the presence of the NOS inhibitor was greater in the
arteries from the pregnant women and could suggest increased synthesis of an endothelium
dependent dilator other than NO or PGI2, potentially an endothelium derived hyperpolarizing
factor. In a later study, the same group (Knock and Poston, 1996) found that pregnancy was
associated with increased relaxation to bradykinin (BK) in small subcutaneous arteries
(Figure 13.3), leading to the suggestion that elements of the signal transduction pathway
for BK were preferentially affected by pregnancy.
In contrast, using arteries from the omental circulation Pascoal et al. (1996) concluded
that neither ACh nor BK mediated relaxation was different in arteries from term pregnant
women and non-pregnant women, although pregnancy was associated with increase in a
novel component of BK mediated relaxation, possibly a hyperpolarising factor (Pascoal et
al., 1996). Another study using the wire myograph has also shown no difference in relaxation
to BK in small myometrial arteries from pregnant women and from non-pregnant women
obtained during hysterectomy (Ashworth et al., 1997) although structurally these arteries
might be expected to be very different from one another. However, Kublickiene et al. (1997a)
have found that if mounted on a perfusion myograph, small myometrial arteries from
pregnant women respond well to ACh and that this relaxation is greater than relaxation to
ACh in omental arteries from term pregnant women, perhaps indicating that enhanced
receptor mediated relaxation may contribute to increased myometrial blood flow in
pregnancy. Taken together these studies show little consenus regarding the role of agonist
stimulated NO synthesis in vasodilation of pregnancy, perhaps because of the different
vascular beds studied.
There is more agreement that flow mediated NO synthesis is raised in the resistance
vasculature in human pregnancy. Cockell and Poston (1997a) have shown that the subcutaneous
arteries from pregnant women demonstrate a remarkably increased response to flow compared
to those from non-pregnant women, which was totally inhibited by L-NAME (Figure 13.4).
Figure 13.3 Concentration-dependent relaxation to bradykinin in isolated subcutaneous fat arteries from
normotensive pregnant (solid circles) and non-pregnant women (open circles). From Knock and Poston, Am J
Obstet Gynecol. 1996, 175, 1668, 1674.
Figure 13.4a,b Percent change in NE-indueed precon.striction in response to incremental increases in flow in
isolated human subcutaneous arteries from normotensive pregnant women (n=10) (a) and normotensive nonpregnant
women (n=10) (b). Black squares indicate the absence of L-NAME; open circles, the presence of L-NAME.
*P<0.01 for pregnant vs pregnant with L-NAME; +P<0.01 for pregnant vs nonpregnant without L-NAME. From
Cockell and Poston, Hypertension, 1997, 30 (part 1), 247–251. With permission from Cockell, and Poston (1997)
Hypertension, 30(part 1), 247–251.
This substantiated earlier work in arteries from pregnant rats (Learmont et al., 1996; Cockell
and Poston, 1997b) showing enhanced flow mediated dilatation, an observation recently
confirmed by Ahokas et al., (1997). Using the same technique, NO mediated responses to
flow have been observed in small myometrial arteries from women at term (Klubickiene
et al., 1997b).
Role of Oestrogens in Pregnancy Associated Alteration in Nitric Oxide Synthesis

From the moment of conception and throughout healthy human pregnancy there is a steady,
but massive (approximately 250 fold) rise in circulating oestradiol levels (Edouard et al.,
1998; Chapman, 1998). Oestrogens are now recognised to cause both endothelium-dependent
and independent vasodilatation.
In vivo studies have suggested that that the acute vasodilator effects of oestrogens are
endothelium dependent (Gilligan, Quyyumi and Cannon, 1994a). 17ß-oestradiol infusion
potentiated the reduction in coronary artery resistance evoked by acetylcholine (ACh) in women
with coronary artery disease, as measured by Doppler ultrasound (Gilligan et al., 1994a).
Endothelium-dependence of the response was indicated as 17ß-oestradiol did not affect
responses to the endothelium independent vasodilators, adenosine and sodium nitroprusside.
17ß-oestradiol also potentiates endothelium dependent rather than independent vasodilatation
in the forearm of post-menopausal women (Gilligan et al., 1994b). The immediate response
was considered too short for gene transcription and protein synthesis, and it was proposed
that 17ß-oestradiol prolonged the half life of NO by scavenging free radicals, which would
otherwise ‘quench’ NO. This antioxidant effect has also been demonstrated in a study in
which ethinylestradiol elevated cGMP in cultured bovine aortic endothelial cells, without
increasing NOS activity, NOS protein or mRNA, but simultaneously was shown to decrease
synthesis of the superoxide radical (Arnal et al., 1996).
Animal studies suggest involvement of the endothelium in the dilatation induced by
longer term exposure to oestrogen (hours to days, i.e. appropriate to that seen during
pregnancy) (Magness and Rosenfeld, 1989). The increase in uterine artery blood flow
following 17ß oestradiol infusion in non-pregnant sheep is blunted by simultaneous local
infusion of L-nitroarginine methyl ester, suggesting oestrogen enhancement of the NO
pathway (Van Buren et al., 1992). The role of NO in this response has been confirmed by
Veille et al., (1996) who have demonstrated that after 3 days of estradiol treatment, enhanced
NO mediated relaxation in the sheep uterine artery is related to greater NOS enzymatic
activity. Moreover, several investigators (Hayashi et al., 1995; Hishikawa et al., 1995 and
MacRitchie et al., 1997) have shown estradiol mediated increases of NO and eNOS protein
in cultured endothelial cells. 17-ß oestradiol pre-treatment of isolated arteries from the
mesenteric vasculature of prepubertal female rats also induces an increase in endothelium
dependent NO mediated flow induced dilatation (Cockell and Poston, 1997b).
Indirect, in vivo evidence from non-pregnant women, also suggests that oestrogens
increase NOS activity. Women with artificially suppressed endogenous oestrogen levels,
given oral oestradiol had raised plasma nitrate levels compared with those measured after
placebo (Ramsay et al., 1995). Consistent with this suggestion, post menopausal women
given transdermal 17ß-oestradiol show enhanced serum levels of nitrite and nitrate (Roselli
et al., 1995).
The mechanism by which oestrogen modulates NO production has not been fully
elucidated, but it is known that there are 11 copies of an incomplete (half palindromic
motif) estrogen response element (ERE) on the endothelial NOS gene 5' flanking
‘promotor’ region (Robinson et al., 1994). Similarly, widely spaced half-palindromic
motifs act synergistically in other genes to form a complete ERE and it is suggested that
the occupied estrogen receptor may activate NOS-III by binding to these regions. A study
of estrogen receptor knock out mice (ERKO) has confirmed a role for estrogen receptors
in NO synthesis but has also produced some unexpected findings. Male homozygous
estrogen receptor knockout mouse (ERKO) had lower basal release of NO than wild type
males. However, female wild type mice had fewer estradiol receptors and lower basal
NO release than the males and there was no difference in ACh induced relaxation between
ERKO and wild type mice, amongst males or females (Rubanyi, Freay, Kauser et al.,
1997). It is now recognised that there are two functionally distinct estrogen receptors,
alpha and beta (Gustafsson, 1997), but which of these is involved in the interrelation
with NOS in the vasculature is unknown.
Acute endothelium-independent vasodilatory effects of 17ß-oestradiol presumably
independent of the nuclear receptor have been observed at supraphysiological levels in isolated
coronary arteries from animals (Jiang et al., 1991) and humans (Chester et al., 1995). The
mechanism of action is in part mediated by antagonism of Ca++ influx through voltage-gated
channels in vascular smooth muscle (Jiang et al., 1992) and in part through potassium channels
(White et al., 1995). It has been suggested that 17ß-oestradiol stimulates cGMP-dependent
phosphorylation of the Ca++ activated potassium channel, leading to potassium efflux,
membrane repolarisation and relaxation of vascular smooth muscle (White et al., 1995).
Prostacyclin in the Maternal Circulation

Although previously a contentious issue, it is now considered that prostacyclin (PGI2 is
more likely to play a role as a local autocoid than to contribute to the lowering of peripheral
resistance in normal pregnancy. Since the half life of PGI2 is so short, evaluation of synthesis
depends on the measurement of stable metabolites. Using a sensitive and specific analytical
method for the detection of the non-enzymic circulating prostacyclin metabolite, 6-oxo-
PGF1α, Barrow et al. (1983) reported that concentrations were too low for PGI2 to function
as a circulating hormone, despite a significant upward trend during pregnancy. This
conclusion was upheld by studies in pregnant animals and women in which infusion of
indomethacin was shown not to affect blood pressure or peripheral resistance (Conrad and
Colpoys, 1986; Sorensen et al., 1992). Urinary excretion of 2,3-dinor-6-keto-PGF1a, the
major systemic enzymic metabolite of PGI2, is also raised early during human pregnancy
and increases with each trimester (Goodman et al., 1982; Fitzgerald et al., 1987). Whilst
reflecting overall PGI2 biosynthesis, measurement of this metabolite in the urine cannot
discriminate between maternal and fetal sources, and may also reflect renal synthesis. In
the sheep, PGI2 biosynthesis seems to be increased preferentially in the uterine circulation
during pregnancy, possibly in response to elevated AII (Magness et al., 1992). Pregnancy
in the ewe is also associated with a dramatic rise in the expression of COX-1 mRNA and
protein in the uterine artery endothelium (Janowiak et al., 1998).
Human Pregnancy is also associated with increased synthesis of the constrictor prostanoid,
thromboxane (TXA2), as assessed by measurement of its stable systemic metabolite 2,3-
dinor-TXB2. TXA2, which in pregnancy seems to be mainly derived from platelets (Fitzgerald
et al., 1987b) increases 3–5 fold during gestation and remains elevated throughout (Fitzgerald
et al., 1987a).
Endothelium Derived Hyperpolarizing Factor (EDHF) in the Maternal

Circulation
Several studies have suggested that prostaglandin and NO independent, but endothelium-
dependent mechanisms of relaxation may be enhanced in human pregnancy (McCarthy et
al., 1994; Pascoal et al., 1996), an observation supported recently in pregnant rat aorta
(Bobadilla et al., 1997) and from our laboratory in pregnant rat mesenteric small arteries
(Gerber et al., 1998). In our study the component of relaxation to acetylcholine which was
insensitive to cyclooxygenase blockade or to inhibition of NO synthase, was greater in
pregnant rats compared with virgin animals and totally inhibited in both groups by slight
membrane depolarization (induced by modest elevation of the potassium concentration in
the organ bath). This is strongly indicative of a role for enhanced synthesis of the putative
endothelium derived hyperpolarizing factor (EDHF) (for review see Garland et al., 1995).
The exact nature of EDHF has proven to be elusive, although it is suggested by some to be
a cytochrome P450 derivative.
Endothelin in the Maternal Circulation

The plasma concentration of ET-1 is not affected by normal pregnancy and is very low or
undetectable in maternal plasma (Wolff et al., 1997). However, ET-1 causes potent
constriction of myometrial arteries from pregnant (Wolff et al., 1993) and non-pregnant
women (Fried and Samuelson, 1991), being more potent than all other constrictors tested
(Wolff et al., 1993). ET-1 was more potent than ET-3 (Wolff et al., 1993) and potentially
plays an important role in the regulation of uteroplacental blood flow.
ENDOTHELIAL CONTROL OF THE FETOPLACENTAL

CIRCULATION
NO in the Fetoplacental Circulation

NO induced dilatation undoubtedly contributes to the maintenance of low resistance and
pressure in the fetoplacental circulation. Infusion of a NOS inhibitor into a perfused human
placental cotyledon leads to an increase in perfusion pressure (Gude et al., 1990). Endothelial
derived NOS has also been identified by immunohistochemical techniques in placental
vessels (Myatt et al., 1993; Buttery et al., 1994) and is strongly expressed in
syncytiotrophoblast, leading to the suggestion that it may play a role in dilatation of the
maternal spiral artery prior to trophoblast invasion. However, the recent demonstration of
lack of NOS expression in invading trophoblast would argue against that proposal (Lyall et
al., 1998). In our laboratory we have used the perfusion myograph to show in vitro flow
mediated release of NO by small fetoplacental arteries (Learmont and Poston, 1996), which
was inhibited by (L-NAME). Interestingly, agonists including ACh, histamine and bradykinin
which are potent endothelium dependent dilators in other circulations through the release
of NO, have little or no effect in small fetoplacental arteries (McCarthy et al., 1994).
The syncytiotrophoblast of normal placenta synthesises corticotrophin releasing hormone
(CRH), as do the fetal membranes (Perkins and Linton, 1995). Maternal serum concentrations
of CRH rise in normal pregnancy and are even higher in abnormal pregnancies (Wolfe et
al., 1988). Despite this, it is as yet not clear whether the increased CRH levels in pregnancy
have any functional significance. CRH may however, play a role in endothelium dependent
dilatation in the placental circulation. In the perfused placental cotyledon preparation, CRH
leads to a reduction in perfusion pressure, mediating vasodilatation through NO release
(Clifton et al., 1995). In contrast, we have observed that CRH has no direct vasodilatory
effect on isolated small fetoplacental arteries (Dixon et al., 1996) suggesting that in the
intact lobule, CRH may have an indirect effect of vascular tone, potentially through release
of NO from syncytiotrophoblast.
Prostacyclin in the Fetoplacental Circulation

Prostacyclin (PGI2) applied directly to isolated stem villous arteries of the fetoplacental
resistance vasculature effects considerable relaxation (Maigaard et al., 1986a). More
PGI2, relative to TXB2 is synthesised in umbilical vessels than maternal vessels (about
50:1 for umbilical veins and 20:1 for umbilical arteries, as compared with 5:1 for
mammary vessels) (Benedetto et al., 1987). A relatively high rate of PGI2 production
in these vessels ensures the well being of the fetus by contributing to a high blood flow
and preventing thrombosis. Furthermore, more PGI2 is produced in the juxtafetal portion
of both umbilical arteries and veins compared with the juxtaplacental umbilical vessels
(Benedetto et al., 1987). The half life of PGI2 in the placenta, however, is very short, as
prostanoids are rapidly enzymatically inactivated in the placenta (Thaler-Dao et al.,
1974). Agonists which stimulate both PGI2 and NO from the vascular endothelium e.g.
histamine, consistently lead to greater NO induced relaxation than prostanoid mediated
relaxation in umbilical arteries, indicating a relatively greater physiological role for
NO than PGI2 (Izumi et al., 1996).
Endothelins in the Fetoplacental Circulation

The endothelins are powerful constrictors of the fetoplacental resistance vasculature
(MacLean, Templeton and McGrath, 1992). Plasma levels of ET-1 in the umbilical artery
are very high at birth (Nisell et al., 1990) and further raised in pregnancies associated
with fetal hypoxia (Ohno et al., 1995). Placental arteries also develop enhanced
sensitivity to endothelin in pregnancies associated with growth retardation (Liu et al.,
1995). Part of the constrictor activity of endothelin in the placental vascualture has
been attributed to synthesis of the constrictor prostanoid TXA2 (Howarth et al., 1995).
ETA and ETB receptors have been identified in the human placental vasculature; ETA
binding sites predominated on veins and arteries in the chorionic plate, while ETB
binding sites were present in high density in stem villi and blood vessels (Rutherford
et al., 1993). Endothelin is present in umbilical arteries and veins at term (Haegerstrand
et al., 1989), but not early in pregnancy (Sexton et al., 1996). ET-1 also occurs at
higher concentrations in fetal compared with maternal blood (Hakkinen et al., 1992;
Malamitsi-Puchner et al., 1995). ET-1 and ET-3 have also been shown to contract human
fetoplacental veins (Mombouli, Wasserstrum and Vanhoutte, 1993). The endothelins
may therefore play a physiological role in the control of the fetoplacental circulation,
particularly at the moment of childbirth when constriction of the umbilical vasculature
would be beneficial. High concentrations in fetal blood could also indicate a role in
fetal development as this peptide is also a growth factor.
PRE-ECLAMPSIA
Pre-eclampsia is a multi-system disorder unique to human pregnancy (Williams and de

Swiet, 1997). It is classically recognised by hypertension, proteinuria and oedema, affects
about three percent of primigravidae and almost invariably occurs after 20 weeks gestation.
Yet these signs conceal the true identity of a disorder that may be recognised prior to the
onset of hypertension and even evolve into eclampsia (convulsions), without hypertension.
Pre-eclampsia remains an important cause of maternal death in Western Europe (Department
of Health, 1998) and the USA (Kaunitz, Hughes, Grimes et al., 1990).
Relative to the vasodilated, plasma expanded state of healthy pregnancy, pre-eclampsia
is a vasoconstricted, plasma contracted condition with evidence of intravascular coagulation.
The fetus is particularly vulnerable from early onset disease and the mother can succumb
following an illness that can include liver haemorrhage and necrosis, acute renal failure,
subendocardial necrosis, microangiopathic haemolysis, a consumptive coagulopathy and
convulsions. The most common causes for maternal death are adult respiratory distress
syndrome (ARDS) and intra-cerebral haemorrhage (Department of Health, 1998).
Whereas healthy maternal endothelium is crucial for the physiological adaptation to
normal pregnancy, the multiple organ failure characteristic of severe pre-eclampsia is
predominantly secondary to widespread endothelial cell dysfunction (Roberts and Redman,
1993). It is not yet known whether the endothelium of women destined to develop
preeclampsia fails to adapt properly, or is damaged by unknown factors during a pre-
eclamptic pregnancy. Prior to the onset of clinically identifiable disease, women destined
to develop pre-eclampsia show evidence of poor placentation (Brosens, Robertson and Dixon,
1972) and high uteroplacental resistance (Bower et al., 1993). How high placental vascular
resistance triggers maternal endothelial dysfunction is still not completely understood.
Abnormalities of the Uterine Vasculature

In normal pregnancy, the most striking change to a maternal artery occurs within the small
spiral arteries of the uterus. These terminal branches of the uterine arteries are the final
pathway by which jets of oxygenated maternal blood are delivered into the intervillous
space of the growing placenta. Normally, during the first trimester, the terminal branches of
the spiral arterioles are invaded by placental cytotrophoblast cells (Brosens, Robertson,
and Dixon, 1967; Pijnenborg et al., 1980). The muscular and elastic components of the
spiral arteriole wall are replaced by a fibrinoid layer of variable thickness, in which
trophoblast cells are embedded. Normally, there is no disruption of the endothelium (Khong,
Sawyer, and Heryet, 1992). Remodelling of the utero-placental vessels is normally complete
by 18 weeks gestation (Matijevic et al., 1995), but in women who develop preeclampsia,
cytotrophoblast invasion of the spiral arteries is incomplete and high resistance vessels that
retain their muscular wall, persist until term (Khong et al., 1986; Zhou et al., 1993). This
incomplete placentation is considered to play an important role in pre-eclampsia, but as it
also occurs in intra-uterine growth retardation, cannot be considered unique to pre-eclampsia.
Furthermore, the endothelium of these abnormal utero-placental arteries is disrupted by
intraluminal endovascular trophoblast (Khong et al., 1992) (see later). Prior to any clinical
symptoms or signs, doppler ultrasound of uterine arteries at 20–24 weeks gestation, can aid
in the identity of women with high resistance vessels, at risk of preeclampsia (Bower, Bewley
and Campbell, 1993).
The Sympathetic Nervous System in Pre-eclampsia

Although this chapter concentrates on the role of the endothelium in pre-eclampsia, some
investigators have suggested that the pathological increase in vascular tone is secondary to
changes in the autonomic nervous system. Women with pre-eclampsia have been found to
have increased sympathetic nerve activity in muscle-nerve fasicles of the peroneal nerve
(Schobel et al., 1996) and higher plasma noradrenaline levels compared with normotensive
women (Manyonda et al., 1998). In this latter study, tyrosine hydroxylase activity and mRNA
levels were greater in placental tissue from pre-eclamptic compared with normotensive
pregnancies. It is proposed that excessive noradrenaline breaks down more triglyceride to
free fatty acids, which are then oxidised to lipid peroxides (Manyonda et al., 1998). The
latter are cytotoxic to endothelial cells (see later). However, systemic blockade of the
autonomic nervous system with tetraethylammonium chloride (TEAC) or high spinal
anaesthesia causes a dramatic fall in blood pressure in normotensive pregnant women, but
is much less effective in women with pre-eclampsia (Assali and Prystowsky, 1950). Despite
the potential non-specificity of this method, this unique study would suggest that the
hypertension of pre-eclampsia is mediated by a factor independent of the autonomic nervous
system.
INVESTIGATION OF ENDOTHELIAL FUNCTION IN PRE-

ECLAMPSIA
Markers of Endothelial Dysfunction

Healthy endothelial cells maintain vascular integrity, prevent platelet adhesion and influence
the tone of underlying vascular smooth muscle. Damaged endothelial cells are unable to
perform these three functions leading to increased capillary permeability, platelet thrombosis
and increased vascular tone (Flavahan and Vanhoutte, 1995). These features are found in
pre-eclampsia and suggest that the maternal syndrome is, at least in part, an endothelial
disorder (Roberts et al., 1989; de Groot and Taylor, 1993). Evidence of endothelial cell
damage prior to clinical manifestation of pre-eclampsia can be demonstrated by the presence
of markers of endothelial cell activation. Specifically, levels of fibronectin (Ballegeer et al.,
1989) and Factor VIII related antigen are elevated (Roberts and Redman, 1993). Furthermore,
women with endothelial cell damage, secondary to pre-existing hypertension or other micro-
vascular disease, have a higher incidence of pre-eclampsia than normotensive women (Ness
and Roberts, 1996).
Morphological evidence of endothelial damage in pre-eclampsia can be seen in the
glomerular capillaries (Figure 13.5) and in the utero-placental arteries (Figure 13.6)—a
vasculopathy known as acute atherosis.
Endothelin in the Maternal Circulation in Pre-eclampsia

Women with pre-eclampsia also have higher plasma endothelin (ET-1) concentrations than
women with a normal pregnancy (Wolff et al., 1996a). This perhaps would be anticipated,
as a marker of the generalized endothelial dysfunction, but nonetheless could contribute to
vasoconstriction. Enhanced sensitivity to endothelin is unlikely to contribute to
vasoconstriction of the maternal vasculature as there is no evidence to suggest an increase in
Figure 13.5 (A) Normal renal glomerulus with patent capillary lumen. (B) Glomerular endotheliosis in a 19 year
old primigravida with pre-eclampsia at 25 weeks gestation. The capillary lumen have been obliterated by swollen
capillary endothelial cells. Photograph kindly provided by Dr. Meryl Griffiths, Department of Pathology, University
College London Medical School.
Figure 13.6 Two placental bed biopsies taken from different women at 31 weeks gestation (haematoxylin and
Eosin stain, both x40). (A) Normal decidual vessel (just below middle of picture, collapsed flat due to lack of
blood). There is a thin layer of media, surrounded by fibrinoid material, (B) Decidual vessel from a woman with
severe pre-eclampsia and intra-uterine growth retardation. There is gross intimal hyperplasia with a much reduced
lumen. In some severe cases of pre-eclampsia, (not seen in this example) the endothelium is replaced by cholesterol-
laden macrophages, hence the term ‘atherosis’. Photographs kindly provided by Prof. Steve Robson, Department
of Fetal Medicine, Newcastle upon Tyne.
responsiveness to ET-1 in pre-eclampsia. Studies in isolated omental (Vedernikov et al.,

1995; Wolff et al., 1996a) and myometrial arteries (Wolff et al., 1996b) have shown similar
responses in arteries from normal and pre-eclamptic women. One report suggests urinary
excretion of ET-1 is reduced in women with pre-eclampsia (Clark et al., 1997) and as
endothelin is natriuretic, could be implicated as a cause of sodium retention.
Nitric Oxide in Pre-eclampsia

The L-arginine-NO pathway is an expected casualty of endothelial cell damage in
preeclampsia. However, probably because of methodological limitations there is no
consensus on whether NOS activity is altered by pre-eclampsia. Most studies have either
shown no change (Cameron et al., 1993; Curtis et al., 1995; Silver et al., 1996), or an
increase (Nobunaga et al., 1996; Smarason et al., 1997) in circulating or urinary NO
metabolites in women with pre-eclampsia, probably reflecting variable nitrate intakes. Only
Seligman et al., 1994 documented lower plasma NOx concentrations in women with pre-
eclampsia compared with normotensive controls. Cameron et al. (1993) and Nobunaga et
al. (1996) documented a correlation between systolic blood pressure and increasing urinary
and plasma concentrations of NOx, respectively. In the latter study, volunteers were starved
for 12–15 hours in an attempt to control for dietary nitrogen.
NOS is competitively inhibited by an endogenous guanidino-substituted arginine
analogue, N GN G dimethylarginine (asymmetric dimethylarginine, ADMA). During
normotensive pregnancy, the plasma concentration of ADMA is lower than non-pregnant
women and those with pre-eclampsia (Pickling et al., 1993). The fall in plasma ADMA
concentration occurs in the first trimester and correlates with the gestational fall in blood
pressure (Holden et al., 1997). Furthermore, plasma ADMA levels are significantly higher
in women with pre-eclampsia than gestationally matched, normotensive controls (Holden
et al., 1997). Consequently, endogenous inhibition of NOS by a specific inhibitor is a possible
mechanism whereby NO production could be reduced in pre-eclampsia.
Nitric Oxide in the Resistance Vasculature in Pre-eclampsia

As outlined above, the lack of conformity amongst the studies investigating agonist
stimulated NO in the resistance vasculature from healthy pregnant women is disappointing.
However, there is much wider agreement that agonist mediated NO synthesis is reduced
in small arteries from women with preeclampsia. As discussed above, pre-eclampsia is
associated with many facets of endothelial cell dysfunction and the abnormal relaxation
to agonists is probably yet another indication of a general endothelial cell defect. In small
arteries from the subcutaneous circulation, sensitivity to both ACh (McCarthy et al., 1994)
and to BK (Knock et al., 1996) is reduced in women with pre-eclampsia. Similarly, in the
omental circulation of women with pre-eclampsia, Pascoal et al., (1998) found relaxation
to ACh was totally absent whilst responses to BK were unaffected when compared to
normotensive controls. In small myometrial arteries from pre-eclamptic women mounted
on a wire myograph, relaxation to BK was completely absent, whereas normotensive
controls relaxed well (Ashworth et al., 1997). In small arteries from the same circulation,
but mounted on a pressurized system, maximal responses to ACh were reduced
(Kublickiene et al., 1998). Whilst largely in agreement as to the loss of endothelium
dependent dilatation, these studies are equivocal regarding the role of NO. Knock et al.,
(1996) and Klubickiene et al. (1998) have suggested that the blunting of responses to
endothelium dependent dilators is due to reduced NO synthesis, but Pascoal et al. (1988)
have suggested that the defect is NO independent. Flow mediated responses, largely NO
dependent, are however, severely blunted in small subcutaneous arteries from women
with preeclampsia (Cockell and Poston, 1997).
Prostanoids in the Maternal Circulation in Pre-eclampsia

In contrast to normal pregnancy, pre-eclampsia is associated with relative underproduction
of PGI2 and over abundance of TXA2 (Fitzgerald et al., 1990). The imbalance between the
synthesis of these prostanoids formed the rationale for investigations of “low dose aspirin”
therapy for prevention of pre-eclampsia. Whereas aspirin in excess of 80 mg/day substantially
inhibits both PGI2 and TXA2, low or intermittent doses lead to preferential inhibition of
TXA2 biosynthesis (Ritter et al., 1989), and could redress the imbalance between these
prostanoids in pre-eclampsia. Selectivity for TXA2 may lie in differential access of aspirin
to platetelet cyclooxygenase in the portal circulation (Pedersen and FitzGerald, 1984) with
presystemic metabolism preventing access of aspirin to the systemic vasculature and placenta
(for review see Dekker and Sibai, 1993) and/or in the differential affinity of aspirin for
cyclooxygenase in platelets (Patrignani et al., 1982). Also platelets, being anuclear, lack
the capacity to regenerate cyclooxygenase, whereas endothelium can regenerate the enzyme
and so maintain PGI2 production (Ritter et al., 1989). The potential benefit of aspirin had
also been highlighted by the early indications of clinical improvement in patients with pre-
eclampsia (Crandon and Isherwood, 1979).
The first, small placebo controlled trials which followed were supportive; the first, a
double blind study, (Wallenburg et al., 1986) showed that aspirin (60 mg/day) given from
28 weeks of gestation significantly lowered the incidence of pre-eclampsia in a group of
‘high risk’ women defined in terms of abnormal presser responsiveness to All. Despite
several susbequent small, but supportive studies, this early promise was not fulfilled. The
largest randomised trial of low dose aspirin to date, the UK based Collaborative Low Dose
Aspirin Study in Pregnancy included 9364 women (CLASP Collaborative Group, 1994).
Overall there was no evidence of any clinically important benefit in women who took 60
mg aspirin rather than placebo. A post hoc analysis of the CLASP data did however show
that the earlier low dose aspirin was started, the greater the protection from pre-eclampsia
(Broughton Pipkin et al., 1996). A subsequent study of prophylactic low dose aspirin (60mg/
day) given to women at high risk of pre-eclampsia from between 13–26 weeks gestation
(arguably too late to be of benefit) conferred no significant benefit compared with high risk
women given placebo (Caritis et al., 1998).
Effect of Sera From Pre-eclamptic Women on Cultured Endothelial Cells

Several experiments have shown changes to different types of cultured endothelial cells
(human umbilical vein (HUVEC), human decidual tissue, animal and fetal cells) following
exposure to plasma or sera from normal pregnant or pre-eclamptic women. Initial reports
tended to show that pre-eclamptic sera contained elements that were cytotoxic to endothelial
cells (Rogers et al., 1988; Tsukimori et al., 1992). However, more recent studies have shown
that pre-eclamptic sera is not cytotoxic to HUVEC (Endresen et al., 1995; Zammit,
Whitworth and Brown, 1996). Methods used included the release from endothelial cells of
51
Chromium, the integrity of cell membranes as judged by the number of trypan blue positive
cells, incorporation of tritiated thymidine and leucine (reflecting DNA and protein synthesis)
and overall cell proliferation (Endresen et al., 1992).
The suggestion that pre-eclampsia was associated with reduced PGI2 synthesis (see above)
prompted investigators to determine whether a blood borne factor or factors could inhibit
endothelial PGI2 production. Surprisingly a study by Baker et al. (1996a) has shown that
plasma from women with pre-eclampsia leads to stimulation of PGI2 release from bovine
coronary microvascular endothelial cells, followed by a subsequent fall. However, a follow
up study from the same group has shown that flow across the endothelial cells (i.e. mechanical
shear stress) negated the difference between the response to normal and pre-eclamptic serum
(Baker et al., 1996b). Another group has reported that endothelial cells isolated from maternal
decidual tissue of women with pre-eclampsia synthesised an equal amount of PGI2 compared
with decidual endothelial cells from normal pregnant women (Gallery et al., 1995a). In
accord with Baker’s first study, incubation with serum from women with preeclampsia, led
to an increase in PGI2 synthesis from maternal (decidual) and fetal (umbilical vein)
endothelial cells and a further increase in PGI2 synthesis if the decidual cells originated
from women with preeclampsia (Gallery et al., 1995b). In contrast, another study has shown
that pre-eclamptic serum did not alter PGI2 synthesis from HUVECs and was not cytotoxic
to endothelial cells after short term incubation (Zammit et al., 1996).
The conflicting results from this group of experiments are likely to represent differences
in experimental method. It is very difficult to draw any other conclusion from studies that
use different concentrations of serum or plasma (ranging from 2%–30%, made up in different
medium), applied to different types of endothelial cells, some from the fetal circulation and
some even from other animals.
Prothrombotic States
Stimulation of the coagulation cascade in response to endothelial cell damage is more likely
in women who have a predisposition to thrombosis. In an uncontrolled study of women
with a history of early severe pre-eclampsia, Dekker et al., 1995 found a significant
association with a subclinical prothrombotic state. Twenty five percent had protein S
deficiency, 18% had hyperhomocysteinaemia and 16% had activated protein C resistance
(Dekker et al., 1995). Similarly, 14 of 158 (8.9%) women with severe pre-eclampsia were
heterozygous for the factor V Leiden mutation as compared with 17 of 403 (4.2%)
normotensive gravid controls (Dizon-Townson et al., 1996). It is possible that during
pregnancy, the normally sub-clinical prothrombotic state causes utero-placental thrombosis
and placental ischaemia which then triggers the maternal syndrome of pre-eclampsia.
All sensitivity in pre-eclampsia

The classical study of Gant et al., (1973) showed that at 22–26 weeks gestation, prior to the
onset of clinically identifiable pre-eclampsia, a group of predominantly black, teenage
American women demonstrated greater sensitivity to an infusion of ANG II compared with
women who did not develop pre-eclampsia. Compatible with underlying endothelial
dysfunction, increased sensitivity to this presser agent was acclaimed as a useful screening
test for pre-eclampsia. However, the positive predictive power of this original study has not
been reproducible in six other populations (Kyle et al., 1995). Indeed, the most
comprehensive study, involving 495 healthy nulliparous women studied at 28 weeks
gestation, revealed a positive predictive value of only, 19% (Kyle et al., 1995). Differences
in the populations studied may explain this disparity, but overall this invasive test, originally
heralded as a predictive test for pre-eclampsia is of no clinical uses in predicting pre-
eclampsia in an otherwise healthy European population.
Nitric Oxide in the Fetoplacental Circulation in Pre-eclampsia

Investigations of the placentae from women with pre-eclampsia using immunohistochemical
methods suggest, in contrast to the majority of studies in the maternal circulation, that the
expression of the endothelial isoform of NOS (eNOS) is increased (Ghabour, Eis, Brockman,
Pollock & Myatt, 1995). Moreover, Lyall et al. (1996) documented an increase in plasma
NO concentrations (measured using the Greiss reaction) in fetal blood from pregnancies
complicated by intrauterine growth restriction. These studies both suggest that NOS could
increase as a compensatory response to poor blood flow. Theoretically, this may occur as a
result of an increase in shear stress arising from elevation of vascular resistance, since shear
is a stimulus to NOS activation. The function of the eNOS identified in the
syncytiotrophoblast of normal placentae has yet to be explained but an intriguing, although
as yet not proven, suggestion is that it may play a role in dilatation of the maternal spiral
arteries during placentation. However, NO is a potent antiaggregatory agent, reduces
leucocyte adhesion and has bactericidal effects. These properties may also be of benefit in
the placenta.
In contrast to the studies showing enhanced NOS in the fetoplacental circulation by
immunohistochemical methods, comparison of human umbilical vein endothelial cells
isolated from normotensive and pre-eclamptic women has shown a similar capacity to
produce NO (Orpana et al., 1996). This was assessed by the accumulation of nitrate and
nitrite in the culture medium and related to the number of viable endothelial cells.
Interestingly the total number of viable endothelial cells and consequently the total NO
production was reduced in the pre-eclamptic group (Orpana et al., 1996). Extrapolation
from larger fetoplacental vessels to the smaller resistance vasculature must however be
made with caution.
Endothelin in the Fetoplacental Circulation in Pre-eclampsia

Elevated concentrations of ET-1 in the umbilical vein in women with pre-eclampsia have
been reported to relate inversely to birth weight (Wolff et al., 1996) and may suggest a
pathological role for endothelin in reduction of uteroplacental blood flow. In contrast to the
maternal circulation, however, a certain refractoriness seems to develop to the elevated
concentrations of ET-1, as reduced sensitivity to the peptide has been observed in umbilical
arteries from preeclamptic pregnancies (Bodelsson et al., 1995). Again, extrapolation to
the resistance vasculature and the control of placental blood flow may be inadvisable, as
many differences exist in responsiveness to vasoactive agents in umbilical and small placental
arteries.
Aetiology of Maternal Endothelial Dysfunction in Pre-eclampsia

How poor placentation and the resultant poor uterine blood flow leads to the maternal
syndrome of pre-eclampsia, characterised by wide spread endothelial cell damage, remains
Figure 13.7 Possible mechanisms for damaged endothelium in pre-eclampsia (see text for details). Evidence
for the following endothelium toxic factors exists; oxidative stress causing lipid peroxidation; cytokine activation
of neutrophils with increased adhesion and toxicity to endothelium; increased markers of endothelium dysfunction
(vWF and fibronectin); decreased vasodilatory response to shear stress (PEI 2); decreased production of
prostacyclin and probably nitric oxide (NO); increased production of plasminogen activator inhibitor (PA-I) to
inhibit fibrinolysis.
uncertain. Over the years there have been many theories, but currently three or four
predominate (Figure 13.7). Substantial evidence now supports a role for oxidative stress.
The imbalance between free radical synthesis and antioxidant capacity may arise from
reduced placental perfusion coupled with dyslipidaemia. Alternative hypotheses include a
role for pro-inflammatory cytokines and pro-thrombotic states. Others suggest that deported
trophoblast fragments may be responsible for maternal endothelial cell damage. Before the
pamogenesis of pre-eclampsia is discussed in more detail, it is worth remembering that
there are other gestational syndromes, that overlap with pre-eclampsia and which have in
common a dysfunctional endothelium.
These include the HELLP syndrome, an acronym for Haemolysis, Elevated Liver enzymes
and Low Platelets, which overlaps with pre-eclampsia in about 20% of cases (Sibai et al.,
1993) and acute fatty liver of pregnancy (AFLP), which is much more rare, but also associated
with pre-eclampsia in about 50% of cases (Mabie, 1992). Although the liver pathology is
different between HELLP (periportal necrosis) and AFLP (central zone necrosis), the extra-
hepatic manifestations are similar and include a microangiopathic haemolytic anaemia
(MAHA). Indeed, pregnancy predisposes to MAHA, including both haemolytic uraemic
syndrome (HUS) and thrombotic thrombocytopaenic purpura (TTP) (Sibai, Kustermann
and Velasco, 1994). Although HELLP and AFLP are both improved by delivery, HUS and
TTP are not. Indeed, most cases of HUS occur immediately post-partum. Patients with
HUS/TTP have platelet rich thrombi occluding arterioles and capillaries, which are
widespread in TTP and predominate in the kidney with HUS. It is possible that the gestational
activation of the endothelium during pregnancy combined with a normally sub-clinical
prothrombotic tendency predisposes to MAHA. In support of this possibility, there have
been several reports of HUS/TTP in association with the anticardiolipin antibody (Kniaz,
Eisenberg, Elrad et al., 1992). Anticardiolipin antibodies have been associated with pre-
eclampsia in some studies (Yasuda, Takakuwa and Tokunaga et al., 1995), but not in others
(D'Anna Scilipati, Leonardi et al., 1997). It is possible that anticardiolipin antibodies are
transiently present during clinically active pre-eclampsia (D.J.Williams, unpublished
observation).
Postpartum acute renal failure is another well recognised condition, associated with
MAHA. The thrombotic occlusion of glomerular capillaries often leads to bilateral renal
cortical necrosis. This used to be a common problem following septic abortion or delivery,
but is now much more rare. There is great similarity between the pathology of postpartum
acute renal failure and the generalised Shwartzman reaction—an experimentally induced
condition in rodents which normally follows two separate injections of endotoxin. Pregnant
animals appear to be primed and require only one injection of endotoxin before developing
glomerular thrombosis (Conger, Falk and Guggenheim, 1981).
Oxidative Stress and Dyslipidaemia
Free radicals may lead directly to endothelial damage through direct cytotoxicity, or indirectly
through the synthesis of lipid peroxides. The evidence for oxidative stress in pre-eclampsia
is substantial. Reduced plasma concentrations of vitamin C (Mikhail et al., 1994), glutathione
peroxidase and of superoxide dismutase (Wang and Walsh, 1996; Poranen et al., 1996)
have been reported. Interestingly, women with pre-eclampsia are often reported to have
higher plasma levels of the anti-oxidant vitamin E, which may be associated with the response
to increased oxidative stress (Uotila, Tuimala and Aarnio, 1993; Schiff et al., 1996). Raised
concentrations of lipid peroxides are also reported, having been evaluated by estimation of
antibodies to oxidized low density lipoprotein (LDL) (Branch, 1994), plasma concentrations
of malondialdehyde (Hubel et al., 1996) and of the isoprostane, 8-epi-PGF2a (Barden et al.,
1996). Isoprostanes are formed by free radical induced oxidation of arachidonic acid and
are increasingly recognized as stable lipid peroxidation products which may accurately
reflect oxidative damage in vivo.
The origin of the reactive oxygen species may lie in the placenta. Placental ischaemia
accelerates trophoblast cell turnover and so increases the concentration of purines which
act as substrate for xanthine dehydrogenase/oxidase (Many, Hubel and Roberts, 1996).
Under hypoxic conditions, xanthine oxidase predominates over xanthine dehydrogenase to
produce urate and a reactive oxygen species. This process could explain why hyperuricaemia
often precedes clinically recognisable pre-eclampsia and occurs prior to any fall in GFR
(Gallery, Hunyor and Gyory, 1979). Leucocyte activation, whether a primary or a secondary
factor in endothelial cell activation will also contribute to oxidative stress through the
generation of superoxide.
The plasma lipid profile is also abnormal in pre-eclampsia, and will also contribute to
the increased generation of lipid oxidation products. Towards the end of normal pregnancy,
maternal plasma levels of cholesterol and triglyceride increase by 50% and 300%,
respectively (Potter and Nestel, 1979). Women with pre-eclampsia have even higher
circulating levels of triglyceride, free fatty acid and total cholesterol (van den Elzen et al.,
1996; Hubel et al., 1996), with a relative increase in LDL cholesterol. Elevated serum levels
of free fatty acids have been shown to increase triglyceride synthesis in cultured endothelial
cells (Endresen, 1992). Under conditions of oxidant stress and hypertriglyceridaemia,
increased amounts of unsaturated fatty acids will be oxidised to lipid peroxides (Chirico et
al., 1993). There is also a qualitative change in LDLs in established pre-eclampsia, with a
shift towards small dense particles (Hubel et al., 1997), a characteristic which predisposes
the LDL particle to oxidation (Chait et al., 1993).
Neutrophil Activation
Neutrophils and platelets are activated in normal pregnancy and further activated in
preeclampsia (Greer et al., 1989; Zemel et al., 1990). Activated neutrophils, adhere to
endothelium and mediate vascular damage by the release of proteases and reactive oxygen
radicals. Neutrophil elastase, a specific marker of neutrophil activation, circulates in higher
concentrations in women with pre-eclampsia compared with normotensive pregnant women
(Greer et al., 1989). Neutrophil adhesion to the endothelium is mediated through the
expression of cell adhesion molecules on the endothelial cell surface. During endothelial
cell activation, expression of certain cell adhesion molecules is increased on both neutrophils
and the endothelium. Neutrophil adhesion is much more marked in women with
preeclampsia, as they have increased expression of certain cell adhesion molecules compared
with healthy normotensive pregnant women (Barden et al., 1997). Specifically, vascular
endothelial cell adhesion molecule (VCAM-1) and E-selectin circulate in higher
concentrations in women with pre-eclampsia than in normotensive pregnant women (Lyall,
Greer, Boswell et al., 1994).
The stimulus to neutrophil activation remains unknown, but pro-inflammatory cytokines
can activate neutrophils and simultaneously increase expression of cell adhesion molecules
on endothelial cells (Lyall and Greer, 1996). Leucocyte TNF-α gene expression and
circulating levels of TNF-α are enhanced in pre-eclamptic patients compared with
normotensive and non-pregnant women (Chen, Wilson, Wang et al., 1996; Kupfermine,
Peaceman, Wigton et al., 1994). Furthermore, in one study the frequency of the TNF1
allele was markedly increased in pre-eclamptic patients (Chen et al., 1996). TNF-α can
generate reactive oxygen species, inhibit NOS, favour synthesising thromboxane A2 over
prostacyclin, change endothelial cells from an anti-haemostatic to a pro-coagulant state
and activate transcription of VCAM-1 (Chen et al., 1996). On the basis of its biological
properties therefore, TNF-α is a strong candidate for mediating endothelial damage in pre-
eclampsia. A unifying hypothesis is that an ischaemic placenta over-produces inflammatory
cytokines, which activate neutrophils and mediate maternal endothelial cell damage and
pre-eclampsia (Conrad and Benyo, 1997).
Trophoblast Deportation
Another theory for the aetiology of pre-eclampsia suggests that cellular material from
the ischaemic placenta may be shed into the maternal circulation and lead to vascular
damage. Trophoblasts are deported from the placenta to the maternal circulation in
normal pregnancy, and in increased numbers in pre-eclampsia (Chua et al., 1991).
However, due to their size, few are likely to reach the arterial circulation. Histological
Figure 13.8 Syncytiotrophoblast microvilli detected in peripheral plasma from normal and pre-eclamptic women.
Bars indicate median values. With permission from Knight et al. (1998), British Journal of Obstetrics and
Gynaecology, 105, 632–640.
evidence has suggested that the trophoblast microvilli on the surface of the placenta from
women with pre-eclampsia are structurally different and fewer in number than those in
normal placentae (Jones and Fox, 1980). This has led to the suggestion that trophoblast
microvilli may be deported as well as the whole cells, a theory that has some strong
support. Knight et al., (1998) detected syncytiotrophoblast microvilli in the plasma of
pregnant women by flow cytometry and fluoroimmunoassay using anti-placental alkaline
phosphatase antibodies for detection. Significantly higher levels were found in women
with pre-eclampsia (Figure 13.8). Concentrations were higher in uterine venous plasma
than in concurrently sampled peripheral venous plasma, thus suggesting placental origin.
The question remains as to whether these microvilli can inflict endothelial cell damage.
Studies from the same group have shown that normal placental microvilli severely alter
proliferation of cultured human umbilical venous endothelial cells (Smarason et al.,
1993) and in an investigation from one of our laboratories we have shown that perfusion
of small sub-cutaneous arteries from pregnant women with microvilli from normal
placenta can lead to a reduction in endothelium dependent relaxation (Cockell et al.,
1997) (Figure 13.9).
Figure 13.9 Transmission electron microscopy (magnification×1250) of cross-section through luminal surface of
maternal small subcutaneous fat artery (a) after perfusion with erythrocyte for 2 hours; (b) after perfusion with
syncytiotrophoblast microvillous membrane vesicles for 2 hours. En=endothelium; SM=smooth muscle; EL=elastic;
ST=syncytiotrophoblast microvillous membrane vesicles. With permission from Cockell, Learmont, Smarason et
al. (1997) British Journal of Obstetrics and Gynaecology, 104, 235–240.
CONCLUSIONS AND THE FUTURE
Despite the work of many investigators working in different fields who endeavour to
understand the mechanism of pathophysiological change during pre-eclampsia, it is still
unclear which are the most important pathogenic factors and which are simply innocent
para-phenomenon. Conversely, attempts to understand the mechanisms of physiological
change during healthy pregnancy have been relatively neglected. Interpretation of studies
comparing pre-eclamptic with normotensive pregnancies would be put in context by
simultaneous analysis of samples from healthy non-gravid controls. Without this information
it is impossible to appreciate how much the physiological baseline has moved during healthy
pregnancy.
Prevention of endothelial disruption during pre-eclampsia is a better strategy than

treatment of its consequences. This may be one reason why attempts to redress the imbalance
between prostacyclin and thromboxane with low dose aspirin have so far proved unhelpful
in reducing the incidence of pre-eclampsis. However, it is still not clear whether a higher
dose of aspirin 150mg daily, given from early in pregnancy (peri-conception) to women at
high risk of pre-eclampsia would be beneficial. Furthermore, until we understand more
about the activity of the L-arginine-NO pathway in pre-eclampsia, trials that supplement
the NOS substrate L-arginine to women at risk of pre-eclampsia, would be premature. Donors
of NO, must also be used with caution as they not only relax vascular smooth muscle, but
have other, non-vascular actions such as ripening of the cervix. Antioxidants acting as free
radical scavengers might be succesful as prophylaxis against endothelial cell dysfunction.
Trials to compare the outcome of pregnancies in women at high risk of pre-eclampsia
supplemented with a diet of antioxidants are already under way.
In conclusion, the endothelium plays a central role in the maternal adaptation to healthy
human pregnancy. The peripheral circulation of the healthy mother is vasodilated and
prothrombotic, but prone to endothelial cell disorders, such as pre-eclampsia—whatever
it’s aetiology. Delivery of the baby is the only definitive way to cure pre-eclampsia, but
other endothelial disorders, such as HUS/TTP, take advantage of the prothrombotic
endothelium and occur more frequently just after delivery.
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Index
ACE gene polymorphism 198 Cyclooxygenase II 231, 236

Adenosine 99 Cyclosporin 211
Adhesion molecules 147, 150 Cysteine 18
ADMA (see also SDMA & L-NMMA) 6, Cytochrome C oxidase 18
16, 159, 213, 263, 115 Cytochrome P450 79
ADP-ases 99 Cytokines 10, 41, 136, 227, 229
Advanced glycosylated end products 174, Cytomegalovirus 149
179
Aldosterone 47, 65 Dahl salt-sensitive rat 112
Anandamide (see also EDHF) 80 DDAH 16, 161
Angiotensin converting enzyme 117, 198 Dimethylarginine dimethylaminohydrolase
Angiotensin converting enzyme inhibitors —see DDAH
183 DOCA salt-sensitive rat 112
Angiotensin II 249, 256 Doppler 193
Angiotensin II receptor antagonists 117,
183 EDCF 113, 66 & Chapter 6
Apoptosis 155, 157 EDHF 177 & Chapter 4
Arachadonic acid 79, 99, 208, 236 in pregnancy 257
Arginine paradox 16, 19, 160 Endothelin and sepsis 235
Arginine transporters 16 Endothelin converting enzyme 34, 48, 208
ATP-sensitive potassium channels 76 in renal disease 266
Endothelin levels 138, 177, 211, 257, 260
Balloon injury 9, 36, 152 Endothelin receptors 35, 36, 43, 50, 206
Big endothelin 214 Endotoxin 227, 268
Bleeding time 9 & Chapter 5
Bosentan 119 & Chapter 2 F2-isoprostanes 170, 268
BQ 788 140 & Chapter 2 Fetoplacental circulation 257
BQ 123 48, 140, 209, 211, 216, Fibrinogen 96
Bradykinin 253 & Chapter 1 Fibronectin 260
Calcium channels 39, 76, 117, 181, 182, G-proteins 38, 159, 161
Calmodulin 11, 80 Gap junctions 76
Carbon monoxide 81 Gene expression 134
Caveoli 12 Gene transfer 157
Chlamydia pneumoniae 149 Gestational diabetes 183
Cholesterol 148, 157 & Chapter 8 Glibenclamide 78
Cholesryramine 158 Glomerulonephritis 210
Chromosomal localisation 10, 12, 31 Glomerulus 260
Conduit artery 3, 133, 194 Glucocorticoids 237
Corticotrophin releasing hormone 257 Growth factors 40, 14
COX—see cyclooxygenase GTP cyclohydrolase 1 232
Coxsackie virus 173 Guanylate cyclase 17, 138, 205
Cyclic GMP levels 250 & Chapter 1
Cyclooxygenase 44, 63, 176, 205, 207, 227 Haemolytic uraemic syndrome 267 283
283
284 Index
Heart failure 70, 131 & Chapter 7 Oestrogens 14, 69, 83, 200, 248, 255
Heme-oxygenase 81 Oxidative stress 267
Herpes virus 149
HMG Co-A reductase inhibitors 158, 183 p53 155
Homocysteine 147 Palmitoylation 14
Hypercholesterolaemia 153, 196 PDGF 14
Perfusion myograph 253
IIb/IIIa receptor 96 Peroxynitrite 18, 102, 155, 162
Indomethacin 66 Phospholipase A2 40
iNOS 10, 157, 207, 211, 233, 238 Phospholipase C 39
Insulin 113, 173 Phospholipase D 40
Integrins 96, 102, 211, 229, 269 Phosphoramidon 48 & Chapter 2
Interleukins 227, 231, 233, 234 Phosphorylation 14
Pituitary 47
Kallikrem-kinin C 237 Plasma levels—see endothelin levels and
nitrate levels
L-754, 142 211 Plasminogen activator inhibitor 249
L-arginine 135, 152, 199, 215 Platelet activating factor 103, 231, 232
L-NMMA 5 & Chapter 1 Platelet adhesion 96
LDL apheresis 158 Platelet-derived growth factor (see PDGF)
LDL cholesterol 152, 182, 196 Polymorphisms 12
Leukocytes 102 Potassium channels 76, 191
Lipid A 227 Preeclampsia 259
Lipooxygenase 99 nitric oxide in 266
Lipoprotein (a) 147, 196 endothelin in 266
LU135252 120 prostanoids in 264
Preproendothelin 31
Matrix 148 Prostacyclin 76, 98, 182, 256,
metalloproteinases 96, 157 Prostaglandin H2 64
MCP-1 150, 160 Prostaglandins 211, 237
Megakaryocytes 95 Protein kinase C 39, 235
Monocytes 148 Proteinuria 176
Muscarinic receptor 4, 135
Myristoylation 14 Receptor complexes 42
Renin angiotensin system 51, 65, 112, 248
N-acetylcysteine 238 Resistance vessels 4, 132, 209, 251
NADH/NADPH oxidase 137, 179, 183
Neurogenic relaxation 4 Salt-sensitive hypertension 112, 115
Neutrophils in pregnancy 269 Sarafotoxin 32 & Chapter 2
NF?B 147, 160 SB 209670 211
Nitrate levels 10, 111, 250 SDMA (see also ADMA & L-NMMA) 6,
Nitric oxide donor 116, 149, 178, 215 16, 161
Nitrosohaemoglobin 18, 103 Sepsis 234
Nitrotyrosine 157, 162 Shear stress 9, 95, 99, 132, 175, 191
and red cells 104
Obesity 178 Sickle cell anaemia 104
Oestrogen receptor knockout 256 Skin microcirculation 47, 175
Index 285
Sodium 209, 248 Thrombospondin 104

Sodium potassium ATPase 210 Thromboxane A2 64, 98, 174
Sodium sensitivity 210 Ticlopidine 96
Spontaneously hypertensive rat 110 Tissue activator inhibitor 249
Stretch channels 7 TNF alpha (see also cytokines) 230, 269
Subcutaneous arteries 253 Transcriptional regulation 14
Substance P 81, 134 Trophoblast 268
Superoxide 18, 136, 162, 178 Type I diabetes 175
Superoxide dismutase 112, 152, 179 Type II diabetes 177
Sympathetic nervous system 51, 260 Tyrosine kinases 40
T cells 149 Ultra sound see Doppler

TAK-044 50, 119, 214
Tetrahydrobiopterin 161 & Chapter 1 Veins 4, 251
Thiazolidonediones 183 Vitamin C 67, 137, 162, 200
Thrombosis 95, 249 Vitamin E 179, 268
Thrombosis in pregnancy 265 von Willebrand factor 104, 174

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Vascular Endothelium in Human Physiology

Centre for Clinical Pharmacology

Clinical Pharmacology Unit and Research Centre

harwood academic publishers

Copyright © 2000 OPA (Overseas Publishers Association) N.V. Published by license

All rights reserved.

British Library Cataloguing in Publication Data

ISBN 0-203-30495-0 Master e-book ISBN

ISBN 0-203-34305-0 (Adobe eReader Format)

2 The Endothelin System: Physiology 31

3 Cyclooxygenase-dependent Endothelium-derived Contracting Factors 63

4 Endothelium-derived Hyperpolarizing Factor 75

5 Regulation of Blood Cell Function by Endothelial Cells 95

6 Endothelial Dysfunction and Hypertension 109

7 Endothelial Control of Vascular Tone in Chronic Heart Failure 131

8 Hypercholesterolemia, Atherosclerosis, and the NO Synthase Pathway 147

9 Endothelial Function in Diabetes Mellitus 173

10 Flow-mediated Dilatation and Cardiovascular Risk 191

11 Endothelial Mediators and Renal Disease 205

12 Infection and Vascular Inflammation 227

13 The Endothelium in Human Pregnancy 247

Sir John Vane

Bagnall, A.J. Cooke, J.P.

Evans, T.W. Mann, G.E.

Haynes, W.B. Noll, G.

Hingorani, A.D. Poston, L.

MacAllister, R.J. Ritter, J.M.

Sudano, I. Vanhoutte, P.M.

Taddei, S. Webb, D.J.

Aroon D.Hingorani and Patrick J.Vallance

NITRIC-OXIDE MEDIATED VASODILATION

The generation of NO from L-arginine can be inhibited competitively by certain guanidino-

Human Vessels In Vitro

the response to acetylcholine is not mediated by endothelial generation of NO in resistance

et al., 1989a), and inhibits the generation of NO as detected by a porphyrinic microsensor

Basal Generation Of Nitric Oxide

circulations of healthy volunteers. Systemic infusion of L-NMMA causes a substantial

Other Actions Of Endothelial Nitric Oxide

MEASUREMENT OF NITRIC OXIDE IN HUMANS

NITRIC OXIDE SYNTHASE IN THE VASCULAR ENDOTHELIUM

The biosynthesis of NO from L-arginine and molecular oxygen is mediated by a family of

Enzymatic Synthesis Of Endothelial Nitric Oxide

Gene Localisation, Structure And Polymorphism

Expression And Subcellular Localisation

caveolin-1 and a proposed caveolin-binding sequence in eNOS in the oxidative domain

Regulation Of Expression And Activity

Post-translational Modification of eNOS

Mechanism of Agonist-induced eNOS Activation

Substrate Concentration-Enzyme Activity Relationship

L-arginine circulates in plasma at a concentration of 50–100 µM and enters cells largely

Endogenous eNOS Inhibitors

MOLECULAR TARGETS FOR ENDOTHELIAL NITRIC OXIDE

Activation of Soluble Guanylate Cyclase

Interaction With Cytochrome C Oxidase

Chemical Reactions of Nitric Oxide

The importance of L-arginine:NO pathway in regulation of vascular tone, and systemic

Alan J.Bagnall1 and David J.Webb2

ENDOTHELIN CONVERTING ENZYME

The isoforms of endothelin exert their physiological effects in a receptor-mediated fashion.

Endothelin Receptor Genes

Table 2.1 Properties of the endothelin receptor subtypes.

Distribution of Endothelin Receptor Genes

Structure of Endothelin Receptors

receptors. These share a structure consisting of an extracellular N-terminal region, seven